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

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 [26] 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 [ 26 ] 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 [27].
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[ 27 ]。

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 [28]. 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[ 28 ]。所有反应均采用三个生物复制品进行。

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. [29] and Fiorella et al. [30], 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）。

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eGFP和hpt 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。

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。

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LcMYB1 、 eGFP 、 hpt II和Vir D基因，但在对照中不存在（图4D ）。

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）。

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 / 2和LcANR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。

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道德批准并同意参与

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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。