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. 2021 Oct 9;17:103. doi: 10.1186/s13007-021-00802-w
。 2021 年 10 月 9 日;17:103。 DOI: 10.1186/s13007-021-00802-w

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis
发根农杆菌介导的毛状根转化作为荔枝基因功能分析的有效系统

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PMCID: PMC8502350  PMID: 34627322
PMCID:PMC8502350 PMID: 34627322

Abstract 抽象的

Background 背景

Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi.
荔枝是热带和亚热带地区重要的经济果树。然而,由于荔枝难以再生和稳定转化,其功能基因组学受到严重阻碍。发根农杆菌介导的毛状根转基因系统提供了研究木本植物功能基因组学的替代方法。然而,荔枝毛状根转基因系统尚未建立。

Results 结果

In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots.
在这项研究中,我们报告了一种快速高效的A 。使用绿色荧光蛋白( GFP )基因作为标记的发根基因介导的中华草共转化系统。 L. chinensis cv. 的叶盘和茎段。 ‘粉红桂味’幼苗能够诱导转基因毛状根。最佳程序涉及使用茎段作为外植体,并由A感染。发根菌株MSU440在光密度(OD 600 )0.7下共培养10 min,共培养3 d,共转化效率为9.33%。此外,毛状根转基因系统还成功验证了荔枝花色苷关键调控基因LcMYB1的功能。 LcMYB1的过度表达产生了红色毛状根,积累了更高含量的花青素、原花青素和黄酮醇。此外,涉及类黄酮途径的基因在红色毛状根中被强烈激活。

Conclusion 结论

We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.
我们首先利用A建立了快速高效的转化系统,用于研究荔枝毛状根的基因功能。通过优化参数获得发根菌株MSU440。以关键花青素调节基因LcMYB1为例,该毛状根转基因系统对荔枝的基因功能分析是有效的。

Keywords: Litchi chinensis, Agrobacterium rhizogenes, Hairy root, Anthocyanin, LcMYB1
关键词:荔枝发根农杆菌,毛状根,花青素,LcMYB1

Background 背景

Litchi (Litchi chinensis Sonn.) is a perennial fruit tree of the Sapindaceae family cultivated in tropical and subtropical zones of the world. It has been cultivated for more than 2300 years in China due to its delicious and nutritive fruits. In the past decade, substantial progress has been experienced into the molecular changes of flower and fruit development in litchi with the development of genetic and genomics tools []. However, gene functional characterization is severely hindered by the lack of an efficient regeneration and transformation protocol for litchi. Litchi is strong recalcitrance to in vitro regeneration. Different explants such as anthers, immature embryos, and leaves has been used to induce callus and somatic embryo, but only few of them regenerate successfully []. The main limitation of somatic embryogenesis is the large number of abnormal somatic embryos produced which cannot germinate or convert into normal plants [].
荔枝( Litchi chinensis Sonn.)是无患子科多年生果树,栽培于世界热带和亚热带地区。由于其果实美味、营养丰富,在中国已有2300多年的栽培历史。在过去的十年中,随着遗传和基因组学工具的发展荔枝花和果实发育的分子变化取得了实质性进展[ ]。然而,由于缺乏有效的荔枝再生和转化方案,基因功能表征受到严重阻碍。荔枝对体外再生有很强的抵抗力。花药、未成熟胚和叶等不同的外植体已被用来诱导组织和体细胞胚,但只有少数能够成功再生[ ]。体细胞胚胎发生的主要限制是产生大量异常体细胞胚胎,这些胚胎无法发芽或转化为正常植物[ ]。

Agrobacterium tumefaciens-mediated transformation has been widely used for plant genetic engineering and the study of gene function []. To date, successful A. tumefaciens-mediated transformation has been reported for several woody fruit crops, such as apple [], peach [], and citrus []. Puchooa [] reported the A. tumefaciens-mediated litchi transformation protocols using leaf as explants, but no plantlets were regenerated from transformed calli. Padilla et al. [] developed an A. tumefaciens-mediated transformation of 'Brewster' litchi with the PISTILLATA cDNA in antisense. In general, the transformation efficiency was low, and an efficient and reproducible transformation protocol is yet to be developed for litchi and other woody fruit crops [].
根癌农杆菌介导的转化已广泛用于植物基因工程和基因功能的研究[ ]。迄今为止,成功A .据报道,根瘤菌介导的转化可用于几种木本水果作物,例如苹果[ ]、桃子[ ]和柑橘[ ]。 Puchooa [ ] 报道了A .使用叶作为外植体的根癌介导的荔枝转化方案,但没有从转化的愈伤组织中再生出幼苗。帕迪拉等人。 [ ]开发了一个A.根瘤菌介导的“布鲁斯特”荔枝与反义PISTILLATA cDNA 的转化。总的来说,转化效率较低,并且尚未针对荔枝和其他木本水果作物开发高效且可重复的转化方案[ ]。

A. rhizogenes-mediated hairy root transformation systems provide an alternative to species recalcitrant to transformation by A. tumefaciens []. Compared to A. tumefaciens-mediated transformation, A. rhizogenes-mediated transformation system is speedy, since transgenic hairy roots grow rapidly without a complex cultured process to regenerated plantlets [, ]. Therefore, A. rhizogenes-mediated transformation constitutes a simple, rapid and efficient method for the production of metabolites and the study of gene function in plants []. In grapevine, hairy roots were used to study the function of VvMybPA1 or VvMybPA2 on the regulation of proanthocyanidin biosynthesis []. Recently, Meng et al. [] developed a simple, fast and efficient A. rhizogenes-mediated transformation for generating stable transgenic roots in living plants to facilitate functional studies in vivo. Subsequently, one-step generation of composite plants with transgenic roots by A. rhizogenes-mediated transformation has been established in peach, cucumber, and soybean []. In sweet potato, A. rhizogenes-mediated in vivo root transgenic system were shown to be a suitable system for the functional characterization of genes involved in salt tolerance []. However, the hairy root system has not been established in litchi. Therefore, an efficient hairy root transformation protocol is needed for rapid characterization of gene function in litchi.
发根农杆菌介导的毛状根转化系统为顽抗根癌农杆菌转化的物种提供了替代方案[ ]。与根癌农杆菌介导的转化相比,发根农杆菌介导的转化系统速度很快,因为转基因毛状根生长迅速,无需复杂的培养过程即可再生植株[ , ]。因此, A .发根基因介导的转化构成了一种简单、快速和有效的方法,用于生产代谢物和研究植物中的基因功能[ ]。在葡萄树中,利用毛状根来研究VvMybPA1VvMybPA2对原花青素生物合成调节的功能[ ]。最近,孟等人。 [ ]开发了一种简单、快速、高效的A.发根基因介导的转化,用于在活体植物中产生稳定的转基因根,以促进体内功能研究。随后, A一步生成了具有转基因根的复合植物。发根基因介导的转化已在 。在甘薯中, A .发根基因介导的体内根转基因系统被证明是用于耐盐相关基因功能表征的合适系统[ ]。然而,荔枝尚未建立起毛状根系。因此,需要一种有效的毛状根转化方案来快速表征荔枝的基因功能。

In this study, we developed a reproducible, rapid and highly efficient A. rhizogenes-mediated hairy root co-transformation system for litchi. We optimized the transformation procedure using Green Fluorescent Protein (GFP) gene as a marker. Finally, we demonstrated the potential of the hairy root transgenic system to enable gene functional studies in litchi using the key transcriptional factor LcMYB1 that regulates anthocyanin synthesis as an example.
在这项研究中,我们开发了一种可重复、快速且高效的A发根基因介导的荔枝毛状根共转化系统。我们使用绿色荧光蛋白(GFP)基因作为标记优化了转化程序。最后,我们以调节花青素合成的关键转录因子LcMYB1为例,证明了毛状根转基因系统在荔枝基因功能研究中的潜力。

Materials and methods 材料和方法

Plant materials and culture media
植物材料和培养基

Seeds of Litchi chinensis Sonn. cv. ‘Fenhongguiwei’ were obtained from South China Agricultural University College, Guangzhou, China. Mature seeds were first washed in running water for 30 min. Thoroughly washed seeds were surface-sterilized with 75% ethanol for 1 min and dipped in sodium hypochlorite solution (1%) for 30 min, then rinsed 5 times with sterile water. Surface sterilized seeds were cultured on 1/2 MS medium [] without sucrose. Culture was maintained at 25 ℃ in full light conditions (3000 lx) on a 16 h/8 h day/night cycle for seed germination.
荔枝的种子。简历。 “粉红桂味”获自中国广州华南农业大学学院。成熟的种子首先用流水清洗30分钟。彻底清洗的种子用75%乙醇表面消毒1分钟,并浸入次氯酸钠溶液(1%)30分钟,然后用无菌水漂洗5次。表面灭菌的种子在不含蔗糖的 1/2 MS 培养基 [ ] 上培养。培养物维持在 25℃、全光照条件下(3000 lx),以 16 小时/8 小时的日/夜周期进行种子萌发。

Agrobacterium strain and binary vectors
农杆菌菌株和二元载体

Agrobacterium rhizogenes strain MSU440 was used to induce transgenic hairy roots in litchi. The coding sequence of LcMYB1 (accession number KY302802) without the termination codon was fused with the green fluorescent protein (eGFP) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter in binary vector pCAMBIA1300 (Fig. 1). Binary vectors pCAMBIA1300-eGFP and pCAMBIA1300-LcMYB1-eGFP were introduced into A. rhizogenes strain by freeze–thaw method [].
使用发根农杆菌菌株MSU440在荔枝中诱导转基因毛状根。将没有终止密码子的LcMYB1 (登录号KY302802 )的编码序列与双元载体pCAMBIA1300中的花椰菜花叶病毒(CaMV)35S启动子控制的绿色荧光蛋白(eGFP)基因融合(图1 )。将二元载体 pCAMBIA1300-eGFP 和 pCAMBIA1300-LcMYB1-eGFP 引入A中。通过冻融法分离发根菌株[ ]。

Fig. 1. 图 1.

Fig. 1

Schematic representation of the T-DNA region of the binary plasmid or pCAMBIA1300-LcMYB1-eGFP (A) and pCAMBIA1300-eGFP (B)
二元质粒或 pCAMBIA1300-LcMYB1-eGFP ( A ) 和 pCAMBIA1300-eGFP ( B ) 的 T-DNA 区域示意图

A. rhizogenes-mediated transformation
A.发根基因介导的转化

A. rhizogenes strain MSU440 harboring binary vector pCAMBIA1300-eGFP or pCAMBIA1300-LcMYB1-eGFP was cultured in 600 μL YEP medium containing 50 mg L−1 kanamycin and 50 mg L−1 streptomycin and incubated overnight at 28 ℃ on a rotary shaker at 200 rpm. After the OD600 value reached to 0.6–0.8, bacterial cells were centrifuged at 5000 rpm for 8 min and re-suspended at different concentration (OD600 = 0.3, 0.5, 0.7, 0.9) in MS liquid medium containing 100 μM acetosyringone (AS). The resulting cell suspension culture was used in transformation.
将携带双元载体 pCAMBIA1300-eGFP 或 pCAMBIA1300-LcMYB1-eGFP 的发根发根菌株MSU440 在含有 50 mg L -1卡那霉素和 50 mg L -1链霉素的 600 μL YEP 培养基中培养,并在 200 ℃ 的旋转摇床上于 28 ℃ 培养过夜。转速。 OD 600值达到0.6-0.8后,将细菌细胞以5000 rpm离心8分钟,并以不同浓度(OD 600 = 0.3、0.5、0.7、0.9)重悬于含有100 μM乙酰丁香酮(AS)的MS液体培养基中。 )。所得细胞悬浮培养物用于转化。

Leaf discs and stem segments from 3- to 5- weeks-old litchi plants were submerged in bacterial solution for different incubation time (10, 20, 30, or 40 min) (infection concentration OD600 = 0.7), followed by removal of excessive liquid on sterile paper. Infected explants were then transferred onto filter papers wetted with the liquid MS medium with 100 μM AS and co-cultivated in the dark for 3, 5, and 7 days. After co-cultivation, infected explants were washed with sterile water and transferred onto MS medium containing 300 mg L−1 timentin and 300 mg L−1 carbenicilin.
将 3 至 5 周龄荔枝植株的叶盘和茎段浸入细菌溶液中不同的培养时间(10、20、30 或 40 分钟)(感染浓度 OD 600 = 0.7),然后去除过量的细菌。无菌纸上的液体。然后将感染的外植体转移到用含有 100 μM AS 的液体 MS 培养基润湿的滤纸上,并在黑暗中共培养 3、5 和 7 天。共培养后,用无菌水洗涤受感染的外植体并转移至含有300 mg L -1特美汀和300 mg L -1羧苄青霉素的MS培养基上。

Fluorescence assay of regenerated hairy roots
再生毛状根的荧光测定

The transgenic hairy roots were detected fluorescence using a stereomicroscope or scanning confocal microscopy (Zeiss, Germany) with filter sets for eGFP (525/50 nm). For histological assay of transgenic hairy roots, 0.5 cm long tip of hairy root was excised, and vertical sections were observed and imaged under a light microscope (DP27; Olympus, Japan).
使用立体显微镜或扫描共聚焦显微镜(蔡司,德国)和 eGFP(525/50 nm)滤光片组检测转基因毛状根的荧光。对于转基因毛状根的组织学测定,切除0.5cm长的毛状根尖端,并在光学显微镜(DP27;奥林巴斯,日本)下观察垂直切片并成像。

RNA isolation and PCR analysis
RNA分离和PCR分析

Hairy root RNA was extracted using RNAprep pure Plant Kit (TIANGEN, China) following the instruction. Then 1 μg total RNA was reverse-transcribed to cDNA using GoScript Reverse Transcription System (Promega, USA). RT-PCR was conducted using 2 × Taq Master Mix (Vazyme, China) following manufacturer’s instruction. Real-time quantitative PCR analysis (RT-qPCR) with SYBR Green Master Mix (Vazyme, China) was run in ABI 7500 Real-Time PCR System (Applied Biosystems, USA). The primers were listed in the Table 1. LcActin (accession number HQ615689) was used as internal control. Relative expression levels of candidate genes were calculated with the formula 2−∆∆Ct []. All reactions were performed with three biological replicates.
使用RNAprep pure Plant Kit(天根,中国)按照说明书提取毛状根RNA。然后使用GoScript逆转录系统(Promega,美国)将1μg总RNA逆转录为cDNA。 RT-PCR 使用 2 × Taq Master Mix(Vazyme,中国)按照制造商的说明进行。使用 SYBR Green Master Mix(Vazyme,中国)在 ABI 7500 实时 PCR 系统(Applied Biosystems,美国)中进行实时定量 PCR 分析(RT-qPCR)。引物列于表1中。 LcActin (登录号HQ615689 )用作内部对照。候选基因的相对表达水平用公式2 −ΔΔCt计算[ ]。所有反应均采用三个生物复制品进行。

Table 1. 表 1.

List of primer sequence 引物序列列表

Gene 基因 Forward primer (5′-3′) 正向引物 (5′-3′) Reverse primer(5′-3′) 反向引物(5′-3′)
For real-time qPCR 用于实时 qPCR
 LcMYB1 ACAGCAGAGACCATTTAGGG TGATGTTTGTCCAAGCAGTTC
 LcPAL GCCAAGCAATTGATTTAAGG CCACTTTGAGCAGATCCTTT
 LcC4H LC4H AGACGACTTGAACCACCGC CCCGAACTCGACTCCCTGT
 LcCHS GACATTGTGGTGGTGGAGGT TATTTAGCGAGACGGAGGAC
 LcCHI CGGAGTTTACTTGGAGGATGT CAGTGACCTTCTCAGAGTATTG
 LcF3H GGTGGATAGATGTGACAAAGGAGT GGTTGTGGGCATTTTGGATAGTA
 LcF3’H LcF3'H GCTCCGTCCATCTCTTCTCC CCGTCTCCGAACACTCTCC
 LcDFR 液晶DFR GGACCCTGAAAACGAAGTAA CACTCCAGCAAGTCTCATCA
 LcANS 兰卡纳斯 AGGAAGTTGGTGGTCTGGAAG CCGTTGCTGAGGATTTCAATGGTG
 LcUFGT 超滤过性转氨酶 GCCACCAGCGGTTCCTAATA ATGCCTCTGCTACTGCTACAATCT
 LcGST 谷胱甘肽转移酶 GAGCATAAGCGTCCTGAGTTTC TCCACGGTCCGCATACTTG
 LcLAR1 TGAGAGTAGAGAAATCCGAATG ATGACCTGTTTGGTAGAGAGAA
 LcLAR2 ATGGCACCGTCAAAGCATAC TTTCCCACAGAGAAGCAAGC
 LcANR AGGGCTATGTTGTTCACACTAC AGCAAAATTGACTGGTGTTG
 LcFLS1 GAGAGAGGTGGTGGACAAGT TTGGAACAAGAACGGTGAG
 LcFLS2 AGCCCATTGAAGGTGTAAAG CTTGGAGCCGTTGGATTA
 LcACT ACCGTATGAGCAAGGAAATCACTG TCGTCGTACTCACCCTTTGAAATC
For analysis of PCR 用于 PCR 分析
 eGFP 绿色荧光蛋白 ATGGTGAGCAAGGGCGAGGAGCTGTTCACC TTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCC
 rol B 罗尔B GCTCTTGCAGTGCTAGATTT GAAGGTGCAAGCTACCTCTC
 hpt II CTATTTCTTTGCCCTCGGACGAGTGCTGGGGCGT ATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGA
 Vir D 病毒D ATGTCGCAAGGCAGTAAG CAAGGAGTCTTTCAGCATG

Analysis of anthocyanin, flavonols and proanthocyanidins content

For anthocyanin quantitation, 50 mg of hairy roots was extracted with a solution of mix methanol, water and concentrated hydrochloric acid (85:12:3) overnight at 4 ℃. Total anthocyanin content was measured using absorbance at 530 nm that have been diluted with pH 1.0 and 5.0 buffers.

For flavonols and proanthocyanidins extraction, hairy roots was extracted using methanol following the method described by Tiberti et al. [] and Fiorella et al. [], respectively. Flavonols and proanthocyanidins content were measured using absorbance at 510 nm and 500 nm, respectively.

Statistical analysis 统计分析

All experiment were conducted with three times. All the statistical analysis were performed using a t-test by SPSS software. Significance was indicated by asterisks * (P < 0.05).
所有实验均进行三次。所有统计分析均采用SPSS软件进行t检验。显着性用星号 * 表示( P < 0.05)。

Results 结果

Transgenic hairy root induction from litchi
荔枝转基因毛状根诱导

Leaves and stems from 4-week-old seedlings were used as transformation explants. Approximately 20 days after infection, calli appeared around the cutting site of leaves and stems (Fig. 2A, C). After about 5 weeks, small hairy roots appeared from the calli and elongated (Fig. 2E, G). The co-transgenic roots were detected by screening for GFP fluorescence signal. GFP expression was initially weak and only observed in a few cells of the calli from leaf and stem explants (Fig. 2B, D). Later, stronger GFP expression was observed in the entire transgenic hairy roots (Fig. 2F, H). To further verify that the co-transgenic nature of hairy roots regenerated, PCR analysis was carried out from 4 randomly selected independent transgenic hairy roots. Bands representing the eGFP and hpt II were detected in the transgenic hairy roots with GFP fluorescence, but not in the control (Fig. 2I). Similarly, the rol B gene responsible for directing hair roots differentiation was also detected in transgenic hairy roots. In addition, Vir D gene required for the T-DNA transfer and processing but located outside of T-DNA region of Ri plasmid was absent in transgenic hairy roots (Fig. 2I), showing that there was no A. rhizogenes contamination. In summary, a successful A. rhizogenes-mediated hairy roots co-transformation system was established in litchi.
4周龄幼苗的叶和茎用作转化外植体。感染后约20天,在叶和茎的切割部位周围出现愈伤组织(图2A 、C)。约5周后,愈伤组织中出现小毛状根并伸长(图2 E、G)。通过筛选 GFP 荧光信号来检测共转基因根。 GFP 表达最初很弱,仅在来自叶和茎外植体的愈伤组织的少数细胞中观察到(图2 B、D)。随后,在整个转基因毛状根中观察到更强的 GFP 表达(图2 F、H)。为了进一步验证再生毛状根的共转基因性质,对随机选择的4个独立转基因毛状根进行PCR分析。在具有 GFP 荧光的转基因毛状根中检测到代表eGFPhpt II的条带,但在对照中未检测到(图2 I)。同样,在转基因毛根中也检测到了负责指导毛根分化的rol B基因。此外,转基因毛状根中不存在T-DNA转移和加工所需但位于Ri质粒T-DNA区域之外的Vir D基因(图2I ),表明不存在A。发根菌污染。综上所述,一个成功的A .建立了荔枝发根基因介导的毛状根共转化系统。

Fig. 2. 图 2.

Fig. 2

A. rhizogenes mediated transformation of litchi. Calli (A) and GFP fluorescence (B) induced from leaf discs. Calli (C) and GFP fluorescence (D) induced from stem fragments. Transgenic hairy root (E) and GFP fluorescence (F) from leaf explants. Transgenic hairy root (G) and GFP fluorescence (H) from stem explants. PCR analysis (I) for eGFP, rol B, hpt II, and Vir D in independent transgenic hairy roots
A.发根基因介导的荔枝转化。由叶盘诱导的愈伤组织 ( A ) 和 GFP 荧光 ( B )。由茎片段诱导的愈伤组织 ( C ) 和 GFP 荧光 ( D )。来自叶外植体的转基因毛状根 ( E ) 和 GFP 荧光 ( F )。来自茎外植体的转基因毛状根 ( G ) 和 GFP 荧光 ( H )。独立转基因毛状根中eGFProl Bhpt IIVir D的PCR分析( I )

Optimization of A. rhizogenes-mediated litchi transformation
优化A .发根菌介导的荔枝转化

In order to optimize the A. rhizogenes co-transformation efficiency, A. rhizogenes concentration, infection time, and duration of co-cultivation were tested. Firstly, various concentrations of A. rhizogenes were compared with the infection time of 10 min and co-cultivation for 3 days. When A. rhizogenes concentration of OD600 value ranged from 0.3 to 0.9, the hairy root regeneration rate of leaf and stem explants ranged from 35 to 48%, but there was no significant difference between leaf and stem explants (Fig. 3A). The highest co-transformation efficiency of leaf and stem were 8.33% and 8.89%, respectively (Fig. 3D). Subsequently, different infection times were tested with the A. rhizogenes concentration of OD600 = 0.7 and co-cultivation for 3 days. The hairy root regeneration rate of leaf and stem explants decreased significantly with the increasing infection times (Fig. 3B). Co-transformation efficiency increased significantly from 5 to 10 min, then decreased significantly. The co-transformation efficiency in 10 min of leaf and stem were 6.67% and 8.89%, respectively (Fig. 3E). Finally, various co-cultivation times were evaluated with the A. rhizogenes concentration of OD600 = 0.7 and infection time for 10 min. The results indicated that the hairy root regeneration rate and co-transformation efficiency of leaf and stem at the 2 days and 3 days co-cultivation duration was significantly higher than that of 4 days and 5 days (Fig. 3C, F). When co-cultivation duration for 3 days, the highest co-transformation efficiency of leaf and stem were 5.56% and 9.33%, respectively. Taken together, these results indicated the optimal infection condition was A. rhizogenes concentration of OD600 = 0.7, infection time for 10 min, and co-cultivation for 3 days.
为了优化A .发根菌共转化效率, A 。测试发根菌浓度、感染时间和共培养持续时间。首先,不同浓度的A发根菌与感染时间10分钟和共培养3天进行比较。当A . OD 600值的发根菌浓度范围为0.3至0.9,叶和茎外植体的毛状根再生率范围为35至48%,但叶和茎外植体之间没有显着差异(图3A )。叶和茎的最高共转化效率分别为8.33%和8.89%(图3D )。随后,用A测试了不同的感染时间。发根菌浓度OD 600 = 0.7并共培养3天。随着感染次数的增加,叶和茎外植体的毛状根再生率显着下降(图3B )。共转化效率在 5 至 10 分钟内显着增加,然后显着下降。叶和茎10分钟内的共转化效率分别为6.67%和8.89%(图3E )。最后,用A评估不同的共培养时间。发根菌浓度OD 600 = 0.7,感染时间10分钟。结果表明,共培养2天和3天时的毛状根再生率和叶茎共转化效率显着高于4天和5天(图3C 、F)。 共培养3 d时,叶和茎的共转化效率最高,分别为5.56%和9.33%。综上所述,这些结果表明最佳感染条件是A发根菌浓度OD 600 = 0.7,感染时间10分钟,共培养3天。

Fig. 3. 图 3.

Fig. 3

Optimization of A. rhizogenes-mediated litchi transformation using leaf and stem as explants. The regeneration rate of hairy roots (AC) and co-transformation efficiency (DF) after infecting different OD600 values, times, and co-cultivation duration of Agrobacterium strain MSU440, respectively. * indicate significant differences (P < 0.05)
优化A .使用叶和茎作为外植体进行发根基因介导的荔枝转化。分别为农杆菌MSU440感染不同OD 600值、次数和共培养时间后的毛状根再生率( AC )和共转化效率( DF )。 * 表示差异显着( P < 0.05)

Gene functional analysis using the hairy root transgenic system
使用毛根转基因系统进行基因功能分析

To further confirm that the hairy root transgenic system is suitable for conducting gene functional analysis, LcMYB1, the key transcription factor that regulates anthocyanin biosynthesis in litchi, was co-transformed in the hairy root system. The coding sequence was cloned into pCAMBIA1300-eGFP vector to generate the C-terminal GFP fusion protein (Fig. 1A). Then the pCAMBIA1300-LcMYB1-eGFP vector was transferred into A. rhizogenes strain MSU440 and used for transformation. Red callus appeared after 3 weeks of overexpressing LcMYB1 in leaf and stem using the hairy root system (Fig. 4A). After 1 more week, red hairy roots were regenerated from the red callus (Fig. 4B, C). PCR analysis revealed that LcMYB1, eGFP, hpt II, and Vir D genes were detected in the red hairy roots, but absent in the control (Fig. 4D).
为了进一步证实毛状根转基因系统适合进行基因功能分析,将调控荔枝花青素生物合成的关键转录因子LcMYB1共转化到毛状根系统中。将编码序列克隆到 pCAMBIA1300-eGFP 载体中,生成 C 端 GFP 融合蛋白(图1 A)。然后将pCAMBIA1300-LcMYB1-eGFP载体转移至发根农杆菌菌株MSU440中并用于转化。使用毛状根系统在叶和茎中过表达LcMYB1 3 周后,出现红色愈伤组织(图4 A)。再过 1 周后,红色愈伤组织再生出红色毛状根(图4 B、C)。 PCR分析显示,在红色毛状根中检测到LcMYB1eGFPhpt IIVir D基因,但在对照中不存在(图4D )。

Fig. 4. 图 4.

Fig. 4

Phenotype and molecular validation of transgenic hairy roots overexpressing LcMYB1 in leaf discs. Callus (A) and transgenic hairy roots (B, C) induced from leaf explants. PCR analysis (D) of LcMYB1, eGFP, rol B, hpt II, and Vir D in independent transgenic hairy roots for validation of overexpressing LcMYB1
叶盘中过表达LcMYB1的转基因毛状根的表型和分子验证。由叶外植体诱导的愈伤组织 ( A ) 和转基因毛状根 ( B , C )。对独立转基因毛状根中的LcMYB1eGFProl Bhpt IIVir D进行PCR分析( D ),以验证LcMYB1的过表达

Upon closer inspection, the cells of red hairy roots were visualized in red by accumulating anthocyanin (Fig. 5A) and all the red cells exhibited stable GFP expression (Fig. 5B). In contrast, anthocyanins and GFP expression were absent in the non-transgenic roots. Quantitative analysis showed that contents of anthocyanins, proanthocyanins, and flavonols were significantly higher in red hairy roots. The relative anthocyanins, proanthocyanins, and flavonols contents in red hairy roots were, respectively, 69, 3, and 2 times greater than the control roots (Fig. 5C).
仔细观察后,红色毛状根的细胞因花青素的积累而呈现红色(图5A ),并且所有红细胞都表现出稳定的GFP表达(图5B )。相反,非转基因根中不存在花青素和 GFP 表达。定量分析表明,红毛根中花青素、原花青素、黄酮醇的含量显着较高。红毛状根中花青素、原花青素和黄酮醇的相对含量分别是对照根的 69 倍、3 倍和 2 倍(图5 C)。

Fig. 5. 图 5.

Fig. 5

Color observation and quantitative analysis of anthocyanins, proanthocyanins, and flavonols contents in red hairy roots. Red coloration (A) was seen in the transgenic hairy roots exhibiting GFP expression (B). C Relative anthocyanin, proanthocyanins, and flavonols contents of wild-type (WT) and transgenic red hairy roots (T)
红毛状根中花青素、原花青素、黄酮醇含量的颜色观察及定量分析在表现出 GFP 表达的转基因毛状根中观察到红色 ( A ) ( B )。 C野生型(WT)和转基因红毛根(T)花青素、原花青素和黄酮醇的相对含量

To confirm the role of LcMYB1, the expression levels of its target genes in the flavonoid pathway, including LcPAL (LITCHI018600), LcC4H (LITCHI031057), Lc4CL (LITCHI002917), LcCHS (LITCHI020852), LcCHI (LITCHI027959), LcF3H (LITCHI006477), LcF3'H (LITCHI023381), LcDFR (LITCHI010261), LcANS (LITCHI022925), LcUFGT (LITCHI002457), LcGST (LITCHI008070), LcFLS1 (LITCHI007338), LcFLS2 (LITCHI011187), LcLAR1 (LITCHI028570), LcLAR2 (LITCHI005474), and LcANR (LITCHI029356) were detected using RT-qPCR. The results indicated that the expression of all these genes were up-regulated (Fig. 6). LcGST4 (accession number KT946768), the gene involved in anthocyanin sequestration in litchi, was up-regulated more than 75281 folds. Additionally, the expression levels of LcFLS1/2 involving in flavonols synthesis and LcLAR1/2 and LcANR involving in proanthocyanidins synthesis were also up-regulated. Taken together, these results confirmed that the transgenic hairy roots system mediated by A. rhizogenes could be used to study anthocyanin metabolism in litchi and offer a simple way to verify gene function in woody plants.
为了证实LcMYB1的作用,检测其在类黄酮通路中的靶基因的表达水平,包括LcPAL (LITCHI018600)、 LcC4H (LITCHI031057)、 Lc4CL (LITCHI002917)、 LcCHS (LITCHI020852)、 LcCHI (LITCHI027959)、 LcF3H (LITCHI006477)、 LcF3'H (LITCHI023381)、 LcDFR (LITCHI010261)、 LcANS (LITCHI022925)、 LcUFGT (LITCHI002457)、 LcGST (LITCHI008070)、 LcFLS1 (LITCHI007338)、 LcFLS2使用 RT-qPCR 检测 (LITCHI011187)、 LcLAR1 (LITCHI028570)、 LcLAR2 (LITCHI005474) 和LcANR (LITCHI029356)。结果表明所有这些基因的表达均上调(图6 )。 LcGST4 (登录号KT946768 )是参与荔枝花青素隔离的基因,上调超过 75281 倍。此外,参与黄酮醇合成的LcFLS1 / 2以及参与原花青素合成的LcLAR1 / 2LcANR的表达水平也上调。综上所述,这些结果证实,由发根农杆菌介导的转基因毛状根系统可用于研究荔枝中的花青素代谢,并为验证木本植物中的基因功能提供一种简单的方法。

Fig. 6. 图 6.

Fig. 6

The expression levels of key enzyme genes involved in flavonoid pathway analysed using RT-qPCR were shown in hotspot map. PAL phenylalanine ammonia lyase, C4H cinnamate-4-hydroxylase, 4CL 4-coumaroyl-coA synthase, CHS chalcone synthase, CHI chalcone-flavanone isomerase, F3H flavanone-3-hydroxylase, F3′H flavonoid-3′-hydroxylase, FLS flavonol synthase, DFR dihydroflavonol-4-reductase, LAR leucoanthocyanidin reductase, ANR anthocyanidin reductase, UFGT UDP-glucose:flavonoid-3-Oglucosyltransferase, GST glutathione-S-transferase
使用 RT-qPCR 分析的黄酮类途径中涉及的关键酶基因的表达水平显示在热点图中。 PAL苯丙氨酸解氨酶、 C4H肉桂酸 4-羟化酶、 4CL 4-香豆酰辅酶A 合酶、 CHS查耳酮合酶、 CHI查尔酮黄烷酮异构酶、 F3H黄烷酮 3-羟化酶、 F3′H类黄酮-3′-羟化酶、 FLS黄酮醇合酶, DFR二氢黄酮醇-4-还原酶, LAR无色花青素还原酶、 ANR花青素还原酶、 UFGT UDP-葡萄糖:类黄酮-3- O葡萄糖基转移酶、 GST谷胱甘肽-S-转移酶

Discussion 讨论

In the last two decades, increasing fruit tree genomic resources like genome sequences were available paving the way to the genetic engineering and molecular breeding of fruit plants for crop improvement []. It will be possible to identify the genes controlling the important horticultural traits. In litchi, transcriptome analysis was used to identify the key genes involving in anthocyanin biosynthesis [, ]. However, their function analysis was usually validated in model plants such as Arabidopsis and tobacco [, , ]. The major reason for this is the lack of litchi transformation system []. An effective transgenic technology will be crucial for genes functional analysis. Thus, our newly developed transgenic hairy roots system mediated by A. rhizogenes will advance functional genomics research in litchi.
在过去的二十年中,不断增加的果树基因组资源(例如基因组序列)为用于作物改良的果树基因工程和分子育种铺平了道路[ ]。鉴定控制重要园艺性状的基因将成为可能。在荔枝中,转录组分析用于鉴定参与花青素生物合成的关键基因[ , ]。 造成这种情况的主要原因是缺乏荔枝转化系统[ ]。有效的转基因技术对于基因功能分析至关重要。因此,我们新开发的由发根农杆菌介导的转基因毛状根系统将推进荔枝的功能基因组学研究。

To date, A. tumefaciens-mediated transformation systems were widely used for functional genomics in plants []. Recently, significant advances have been made in optimizing the genetic transformation of fruit trees [, ]. In litchi, only one report was successful in developing transgenic plants using A. tumefaciens-mediated method []. In this report, only gene expression levels of the four transgenic lines were analyzed without any phenotype analysis, as it needs 7–8 years (typical juvenile period for litchi) to obtain mature plants []. Therefore, A. tumefaciens-mediated transformation is time-consuming, recalcitrant nature and not efficient enough to allow the high-throughput for functional genomic research []. Besides A. tumefaciens, A. rhizogenes is also utilized on functional studies of genes, especially those involved in secondary metabolism and responses to environmental stresses []. Here, A. rhizogenes-mediated transformation was established in litchi using leaf and stem as explants. Similar report also showed that hypocotyl, leaf and shoot were suitable for A. rhizogenes-mediated transformation in peach [].
迄今为止,根癌农杆菌介导的转化系统已广泛用于植物的功能基因组学[ ]。最近,在优化果树遗传转化方面取得了重大进展[ , ]。在荔枝中,只有一份报告成功地利用A .根癌介导的方法[ ]。在本报告中,仅分析了四个转基因品系的基因表达水平,而没有进行任何表型分析,因为它需要7-8年(荔枝的典型幼年期)才能获得成熟植株[ ]。因此, A .根瘤菌介导的转化是耗时的、顽固的,并且效率不够高,无法实现功能基因组研究的高通量[ ]。除了A。 tumefaciens , A. rhizogenes也用于基因的功能研究,特别是那些涉及次生代谢和对环境胁迫反应的基因 [ ]。这里,使用叶和茎作为外植体在荔枝中建立发根农杆菌介导的转化。类似的报告还表明,桃子的下胚轴、叶和芽适合发根农杆菌介导的转化[ ]。

Factors including Agrobacterium concentration, infection time, and co-cultivation time affect T-DNA delivery and its integration into the plant genome [, ]. In the present study, the co-transformation efficiency for leaf and stem explants were higher after they were co-cultivated for 2 or 3 days. With a longer period of 4 or 5 d, A. rhizogenes may overgrow, leading to explant cell damage, consequently, to low transformation efficiency []. There was no significantly difference of co-transformation efficiency for leaf and stem explants in litchi. Liu et al. [] reported that the A. rhizogenes transformation efficiency varied with the type of explant of Arachis hypogaea and was highest with petioles.
农杆菌浓度、感染时间和共培养时间等因素会影响 T-DNA 的传递及其与植物基因组的整合 [ , ]。在本研究中,叶和茎外植体共培养2或3天后,共转化效率较高。如果时间较长,如4或5天,发根农杆菌可能会过度生长,导致外植体细胞损伤,从而导致转化效率低下[ ]。荔枝叶和茎外植体的共转化效率没有显着差异。刘等人。 [ ]报道发根农杆菌转化效率随花生外植体类型的不同而变化,其中以叶柄最高。

So far, transgenic hairy roots system mediated by A. rhizogenes has already been widely applied for many purposes including metabolism, root biology, and stress response [, , ]. Here, we used the established transgenic hairy roots system to study the gene function of LcMYB1 which regulates anthocyanin biosynthesis in litchi. The result indicated that overexpression of LcMYB1 could induce anthocyanin accumulation and produce red hairy roots. Previously, anthocyanins were accumulated in the tobacco hairy roots overexpressing LcMYB1 []. RT-qPCR showed that LcMYB1 could induce the expression of structural genes involved in anthocyanin biosynthetic in hairy roots of litchi. Therefore, transgenic hairy roots system was validated as an effective overexpression technique to study gene function in litchi. In addition, transgenic root system allows for silencing techniques or gene editing to be applied to plants that are recalcitrance to in vitro regeneration [, ]. These techniques in litchi need to be further explored.
,由发根农杆菌介导的转基因毛状根系统已广泛应用于 。在此,我们利用已建立的转基因毛状根系统来研究调控荔枝花青素生物合成的LcMYB1基因的功能。结果表明, LcMYB1的过表达可以诱导花青素积累并产生红色毛状根。此前,花青素在过表达LcMYB1的烟草毛状根中积累[ ]。 RT-qPCR表明, LcMYB1可以诱导荔枝毛状根中参与花青素生物合成的结构基因的表达。因此,转基因毛状根系统被验证为研究荔枝基因功能的有效过表达技术。此外,转基因根系统允许将沉默技术或基因编辑应用于难于体外再生的植物[ , ]。荔枝的这些技术还有待进一步探索。

Conclusion 结论

Here, we first established a rapid and efficient root transgenic system for litchi. The optimal parameters were infection by A. rhizogenes strain MSU440 at OD600 of 0.7 for 10 min and co-cultivation for 3 days. We validated this transformation system for study gene function in transgenic hairy roots by producing transgenic roots overexpressing LcMYB1, the key transcription factor that regulates anthocyanin biosynthesis in litchi. Transgenic roots demonstrated red color and increased flavonoid contents and displayed upregulation of flavonoid-related genes. These results will help to provide a simple and rapid method for gene function analysis in litchi.
在这里,我们首先建立了快速高效的荔枝根系转基因系统。最佳参数是发根农杆菌菌株MSU440以OD 600 0.7感染10分钟并共培养3天。我们通过产生过度表达LcMYB1 (调节荔枝花青素生物合成的关键转录因子)的转基因根来验证该转化系统用于研究转基因毛状根中的基因功能。转基因根呈红色,类黄酮含量增加,并显示类黄酮相关基因的上调。这些结果将为荔枝基因功能分析提供一种简单、快速的方法。

Acknowledgements 致谢

Not applicable. 不适用。

Authors' contributions 作者的贡献

JZ carried out conception of the research, and wrote the manuscript. YQ, DW and JZ performed the experiments. JF, ZZ, and YQ gave the critical suggestion of this study and revised the manuscript. GH supervised the entire study. All authors read and approved the final manuscript.
JZ进行了研究构思,并撰写了手稿。 YQ、DW 和 JZ 进行了实验。 JF、ZZ和YQ对本研究提出了批评性建议并修改了手稿。 GH 监督了整个研究。所有作者阅读并批准了最终手稿。

Funding 资金

This work was funded by the National Key Research and Development Program (No. 2019FYD1000900), the National Natural Science Fund of China (No. 31872066), China Litchi and Longan Industry Technology Research System (No. CARS-32-05), the Science and Technology Planning Project of Guangzhou (No. 202103000057), and the Science and Technology Talents and Platform Plan of Yunnan Province (No. 202104AC100001-B04).
该工作得到了国家重点研发计划(No. 2019FYD1000900)、国家自然科学基金(No. 31872066)、中国荔枝龙眼产业技术研究体系(No. CARS-32-05)、广州市科技计划项目(202103000057)、云南省科技人才与平台计划(编号202104AC100001-B04)。

Availability of data and materials
数据和材料的可用性

Not applicable. 不适用。

Declarations 声明

Ethics approval and consent to participate
道德批准并同意参与

Not applicable. 不适用。

Consent for publication 同意发表

Not applicable. 不适用。

Competing interests 利益竞争

The authors have no competing interests to declare.
作者没有需要声明的竞争利益。

Footnotes 脚注

Publisher's Note 出版商备注

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
施普林格·自然对于已出版的地图和机构隶属关系中的管辖权主张保持中立。

Contributor Information 贡献者信息

Guibing Hu, Email: guibing@scau.edu.cn.
胡桂兵,Email:guibing@scau.edu.cn。

Jietang Zhao, Email: jtzhao@scau.edu.cn.
赵杰堂,邮箱:jtzhao@scau.edu.cn。

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