木聚糖水解酶基因RcXYNC调控月季黑斑病抗性的研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0766

摘要/Abstract

摘要：

目的由蔷薇盘二孢（Marssonina rosae）侵染引起的黑斑病是庭院栽培月季最常见的真菌性病害，严重影响了月季的观赏价值和经济价值。前期发现木聚糖水解酶RcXYNC在易感和高抗月季株系中差异表达，但其调控机制尚不清晰。拟探究该基因功能，为月季黑斑病抗性机制解析提供依据。方法以月季易感株系R12-26和高抗株系R13-54为材料，克隆RcXYNC的编码序列及启动子序列；利用荧光定量PCR技术检测该基因在蔷薇盘二孢侵染月季不同时期的相对表达量；采用瞬时过表达和VIGS技术对RcXYNC进行功能分析；测定过表达/沉默处理后叶片过氧化氢累积，水杨酸、茉莉酸/乙烯通路抗性标志基因的表达水平；利用酵母双杂交技术挖掘与RcXYNC互作的目标蛋白。结果 RcXYNC的编码序列及启动子序列在高抗/易感株系中一致，均含有1个Glyco-10保守结构域，存在胁迫响应元件。瞬时过表达RcXYNC后，月季叶片的黑斑面积均显著增大；沉默RcXYNC后，黑斑面积均显著减小。瞬时过表达或沉默RcXYNC后，月季叶片中水杨酸、茉莉酸/乙烯通路的大部分抗性标志基因的表达显著上升，其他基因无显著变化。挖掘到20个与RcXYNC互作的蛋白，大多参与植物细胞代谢过程。结论 RcXYNC是月季黑斑病抗性的负调控因子，可能通过影响水杨酸、茉莉酸/乙烯信号通路降低月季的黑斑病抗性。

关键词: 月季, 黑斑病, 蔷薇盘二孢, 细胞壁, 生物胁迫, RcXYNC

Abstract:

Objective The black spot disease caused by Marssonina rosae is the most prevalent fungal disease of garden roses, severely impacting the ornamental and economic value. Previous studies have shown that the xylan hydrolase gene RcXYNC was differentially expressed in susceptible and highly resistant rose strains; however, the regulatory mechanism remains unclear. The objective of our research is to investigate the function of RcXYNC and its resistance mechanism to black spot disease. Method Firstly, the coding and promoter sequences of RcXYNC were cloned from the susceptible rose strain R12-26 and highly resistant strain R13-54. The relative expression of RcXYNC of M. rosae-infected leaves at different stageswas detected by quantitative real-time PCR (RT-qPCR). Then, the function of RcXYNC was analyzed by transient overexpression and virus-induced gene silencing (VIGS) and the H₂O₂ accumulation, transcription levels of defense-related genes in the salicylic acid and jasmonic acid/ethylene signaling pathways were measured. Finally, the target proteins interacting with RcXYNC were identified by yeast two-hybrid system. Result The coding and promoter sequences of RcXYNC were consistent in highly resistant and susceptible strains, both containing a conserved domain of Glyco-10 and stress responsive elements. Transient overexpression of RcXYNC led to a significant increase in black spot areas on rose leaves while silencing of RcXYNC resulted in a significant reduction in black spot areas. Transient overexpression or silencing of RcXYNC significantly increased the expressions of most salicylic acid and jasmonic acid/ethylene signaling pathway defense-related genes in rose leaves, while the rest genes showed no significant changes. Total 20 proteins interacting with RcXYNC were discovered, and most of them were involved in plant cell metabolism processes. Conclusion RcXYNC is a negative regulator to defense against M. rosae, potentially reducing resistance of roses to black spot disease by affecting the salicylic acid and jasmonic acid/ethylene signaling pathways.

Key words: rose, black spot disease, Marssonina rosae, cell wall, biological stress, RcXYNC

苏琳, 戚雨岑, 杨艺, 程和丰, 纪思雨, 刘婷, 程堂仁, 王佳, 张启翔, 潘会堂. 木聚糖水解酶基因RcXYNC调控月季黑斑病抗性的研究[J]. 生物技术通报, 2025, 41(4): 211-226.

SU Lin, QI Yu-cen, YANG Yi, CHENG He-feng, JI Si-yu, LIU Ting, CHENG Tang-ren, WANG Jia, ZHANG Qi-xiang, PAN Hui-tang. Regulating the Resistance to Black Spot Disease by Xylanase Hydrolase Gene RcXYNC in Roses[J]. Biotechnology Bulletin, 2025, 41(4): 211-226.

图/表 9

表1 与RcXYNC互作的候选蛋白

Table 1 Candidate proteins potentially interacting with RcXYNC

序号 Serial number	编号 Number	NCBI序列号 NCBI accession	蛋白注释 Protein annotation
X1	RC0G0005100	XM_024324446.2	月季RNA结合蛋白MSI同源蛋白2 Rosa chinensis RNA-binding protein MSI homolog 2
X2	RC0G0013700	XM_024326448.2	月季叶绿体a-b结合蛋白151 Rosa chinensis chlorophyll a-b binding protein 151
X3	RC0G0171100	XM_024335925.2	月季60S核糖体蛋白L6 Rosa chinensis 60S ribosomal protein L6
X4	RC1G0312200	XM_024317775.2	月季RING蛋白1a Rosa chinensis RING-box protein 1a
X5	RC1G0321200	XM_040516491.1	月季细胞分裂蛋白FtsY同源蛋白 Rosa chinensis cell division protein FtsY homolog
X6	RC1G0417300	XM_024334311.2	月季叶绿体a-b结合蛋白6A Rosa chinensis chlorophyll a-b binding protein 6A
X7	RC1G0487500	XM_024308438.1	月季未知蛋白 Rosa chinensis uncharacterized
X8	RC2G0046000	XM_024329020.2	月季GTP结合核蛋白Ran-3 Rosa chinensis GTP-binding nuclear protein Ran-3
X9	RC2G0102900	XM_040515202.1	月季20 kD分子伴侣蛋白 Rosa chinensis 20 kD chaperonin
X10	RC2G0170900	XM_024327498.2	月季放氧增强蛋白1 Rosa chinensis oxygen-evolving enhancer protein 1
X11	RC2G0252900	XM_024330212.2	月季组蛋白H3.3 Rosa chinensis histone H3.3
X12	RC2G0273300	XM_024330385.1	月季PLAT结构域蛋白3 Rosa chinensis PLAT domain-containing protein 3
X13	RC2G0334800	XM_024326224.2	月季转录因子TGA2.3 Rosa chinensis transcription factor TGA2.3
X14	RC2G0337100	XM_024329362.2	月季核酮糖二磷酸羧化酶小链 Rosa chinensis ribulose bisphosphate carboxylase small chain
X15	RC2G0436200	XM_024323609.2	月季磷脂酶 Rosa chinensis phospholipase D alpha 1
X16	RC2G0583300	XM_024324653.2	月季蛋白翻译因子SUI1同源蛋白2 Rosa chinensis protein translation factor SUI1 homolog 2
X17	RC2G0682000	XM_024324666.2	月季50S核糖体蛋白L27 Rosa chinensis 50S ribosomal protein L27
X18	RC3G0212600	XR_002933950.2	月季未知蛋白 Rosa chinensis uncharacterized
X19	RC3G0287600	XM_024331350.2	月季叶绿体a-b结合蛋白CP29.2 Rosa chinensis chlorophyll a-b binding protein CP29.2
X20	RC3G0321300	XM_024335622.2	月季未知蛋白 Rosa chinensis uncharacterized
X21	RC4G0025300	XM_024338987.2	月季3-酮酰基辅酶A合成酶11 Rosa chinensis 3-ketoacyl-CoA synthase 11
X22	RC4G0451500	XM_024341654.2	月季泛素40S核糖体蛋白S27a Rosa chinensis ubiquitin-40S ribosomal protein S27a
X23	RC4G0478900	XM_024338228.2	月季转录因子MYB1R1 Rosa chinensis transcription factor MYB1R1
X24	RC5G0099800	XM_024343555.2	月季14-3-3型D蛋白 Rosa chinensis 14-3-3-like protein D
X25	RC5G0122400	XM_024305557.2	月季叶绿体a-b结合蛋白LHCII 1型 Rosa chinensis chlorophyll a-b binding protein of LHCII type 1
X26	RC5G0122500	XM_024302937.2	月季叶绿体a-b结合蛋白LHCII 1型 Rosa chinensis chlorophyll a-b binding protein of LHCII type 1
X27	RC5G0175700	XM_024305213.2	月季光系统I反应中心亚基Ⅲ Rosa chinensis photosystem I reaction center subunit Ⅲ
X28	RC5G0230100	XM_024301146.2	月季翻译延长因子2 Rosa chinensis elongation factor 2
X29	RC5G0317100	XM_024342352.2	月季叶绿体a-b结合蛋白3 Rosa chinensis chlorophyll a-b binding protein 3
X30	RC5G0380400	XM_024301028.2	月季细胞分裂蛋白FtsZ同源蛋白1 Rosa chinensis cell division protein FtsZ homolog 1
X31	RC5G0648200	XM_024342978.2	月季果糖二磷酸醛缩酶 Rosa chinensis fructose-bisphosphate aldolase
X32	RC6G0082300	XR_005801236.1	月季未知蛋白 Rosa chinensis uncharacterized
X33	RC6G0168800	XM_024312327.2	月季26S蛋白酶体非ATPase调节亚基14同源蛋白 Rosa chinensis 26S proteasome non-ATPase regulatory subunit 14 homolog
X34	RC6G0391500	XM_024307241.2	月季光系统Ⅱ反应中心W蛋白 Rosa chinensis photosystem Ⅱ reaction center W protein
X35	RC6G0394400	XM_024305667.2	月季MPBQ转移酶 Rosa chinensis 2-methyl-6-phytyl-1， 4-hydroquinone methyltransferase
X36	RC6G0457600	XM_024306392.2	月季40S核糖体蛋白S11 Rosa chinensis 40S ribosomal protein S11
X37	RC6G0524600	XM_024312257.2	月季硫氧还蛋白CDSP32 Rosa chinensis thioredoxin-like protein CDSP32
X38	RC7G0139800	XM_024312740.2	月季28 kD核糖核蛋白 Rosa chinensis 28 kD ribonucleoprotein
X39	RC7G0322800	XM_024317397.2	月季磷酸核酮糖激酶 Rosa chinensis phosphoribulokinase
X40	RC7G0482200	XM_024312769.2	月季60S核糖体蛋白L13a-4 Rosa chinensis 60S ribosomal protein L13a-4
X41	RC7G0520700	XM_024313678.2	月季表皮蜡质合成相关蛋白2 Rosa chinensis protein ECERIFERUM 2

表1 与RcXYNC互作的候选蛋白

Table 1 Candidate proteins potentially interacting with RcXYNC

序号 Serial number	编号 Number	NCBI序列号 NCBI accession	蛋白注释 Protein annotation
X1	RC0G0005100	XM_024324446.2	月季RNA结合蛋白MSI同源蛋白2 Rosa chinensis RNA-binding protein MSI homolog 2
X2	RC0G0013700	XM_024326448.2	月季叶绿体a-b结合蛋白151 Rosa chinensis chlorophyll a-b binding protein 151
X3	RC0G0171100	XM_024335925.2	月季60S核糖体蛋白L6 Rosa chinensis 60S ribosomal protein L6
X4	RC1G0312200	XM_024317775.2	月季RING蛋白1a Rosa chinensis RING-box protein 1a
X5	RC1G0321200	XM_040516491.1	月季细胞分裂蛋白FtsY同源蛋白 Rosa chinensis cell division protein FtsY homolog
X6	RC1G0417300	XM_024334311.2	月季叶绿体a-b结合蛋白6A Rosa chinensis chlorophyll a-b binding protein 6A
X7	RC1G0487500	XM_024308438.1	月季未知蛋白 Rosa chinensis uncharacterized
X8	RC2G0046000	XM_024329020.2	月季GTP结合核蛋白Ran-3 Rosa chinensis GTP-binding nuclear protein Ran-3
X9	RC2G0102900	XM_040515202.1	月季20 kD分子伴侣蛋白 Rosa chinensis 20 kD chaperonin
X10	RC2G0170900	XM_024327498.2	月季放氧增强蛋白1 Rosa chinensis oxygen-evolving enhancer protein 1
X11	RC2G0252900	XM_024330212.2	月季组蛋白H3.3 Rosa chinensis histone H3.3
X12	RC2G0273300	XM_024330385.1	月季PLAT结构域蛋白3 Rosa chinensis PLAT domain-containing protein 3
X13	RC2G0334800	XM_024326224.2	月季转录因子TGA2.3 Rosa chinensis transcription factor TGA2.3
X14	RC2G0337100	XM_024329362.2	月季核酮糖二磷酸羧化酶小链 Rosa chinensis ribulose bisphosphate carboxylase small chain
X15	RC2G0436200	XM_024323609.2	月季磷脂酶 Rosa chinensis phospholipase D alpha 1
X16	RC2G0583300	XM_024324653.2	月季蛋白翻译因子SUI1同源蛋白2 Rosa chinensis protein translation factor SUI1 homolog 2
X17	RC2G0682000	XM_024324666.2	月季50S核糖体蛋白L27 Rosa chinensis 50S ribosomal protein L27
X18	RC3G0212600	XR_002933950.2	月季未知蛋白 Rosa chinensis uncharacterized
X19	RC3G0287600	XM_024331350.2	月季叶绿体a-b结合蛋白CP29.2 Rosa chinensis chlorophyll a-b binding protein CP29.2
X20	RC3G0321300	XM_024335622.2	月季未知蛋白 Rosa chinensis uncharacterized
X21	RC4G0025300	XM_024338987.2	月季3-酮酰基辅酶A合成酶11 Rosa chinensis 3-ketoacyl-CoA synthase 11
X22	RC4G0451500	XM_024341654.2	月季泛素40S核糖体蛋白S27a Rosa chinensis ubiquitin-40S ribosomal protein S27a
X23	RC4G0478900	XM_024338228.2	月季转录因子MYB1R1 Rosa chinensis transcription factor MYB1R1
X24	RC5G0099800	XM_024343555.2	月季14-3-3型D蛋白 Rosa chinensis 14-3-3-like protein D
X25	RC5G0122400	XM_024305557.2	月季叶绿体a-b结合蛋白LHCII 1型 Rosa chinensis chlorophyll a-b binding protein of LHCII type 1
X26	RC5G0122500	XM_024302937.2	月季叶绿体a-b结合蛋白LHCII 1型 Rosa chinensis chlorophyll a-b binding protein of LHCII type 1
X27	RC5G0175700	XM_024305213.2	月季光系统I反应中心亚基Ⅲ Rosa chinensis photosystem I reaction center subunit Ⅲ
X28	RC5G0230100	XM_024301146.2	月季翻译延长因子2 Rosa chinensis elongation factor 2
X29	RC5G0317100	XM_024342352.2	月季叶绿体a-b结合蛋白3 Rosa chinensis chlorophyll a-b binding protein 3
X30	RC5G0380400	XM_024301028.2	月季细胞分裂蛋白FtsZ同源蛋白1 Rosa chinensis cell division protein FtsZ homolog 1
X31	RC5G0648200	XM_024342978.2	月季果糖二磷酸醛缩酶 Rosa chinensis fructose-bisphosphate aldolase
X32	RC6G0082300	XR_005801236.1	月季未知蛋白 Rosa chinensis uncharacterized
X33	RC6G0168800	XM_024312327.2	月季26S蛋白酶体非ATPase调节亚基14同源蛋白 Rosa chinensis 26S proteasome non-ATPase regulatory subunit 14 homolog
X34	RC6G0391500	XM_024307241.2	月季光系统Ⅱ反应中心W蛋白 Rosa chinensis photosystem Ⅱ reaction center W protein
X35	RC6G0394400	XM_024305667.2	月季MPBQ转移酶 Rosa chinensis 2-methyl-6-phytyl-1， 4-hydroquinone methyltransferase
X36	RC6G0457600	XM_024306392.2	月季40S核糖体蛋白S11 Rosa chinensis 40S ribosomal protein S11
X37	RC6G0524600	XM_024312257.2	月季硫氧还蛋白CDSP32 Rosa chinensis thioredoxin-like protein CDSP32
X38	RC7G0139800	XM_024312740.2	月季28 kD核糖核蛋白 Rosa chinensis 28 kD ribonucleoprotein
X39	RC7G0322800	XM_024317397.2	月季磷酸核酮糖激酶 Rosa chinensis phosphoribulokinase
X40	RC7G0482200	XM_024312769.2	月季60S核糖体蛋白L13a-4 Rosa chinensis 60S ribosomal protein L13a-4
X41	RC7G0520700	XM_024313678.2	月季表皮蜡质合成相关蛋白2 Rosa chinensis protein ECERIFERUM 2

图1 RcXYNC基因的生物信息学特征A：基因编码序列克隆凝胶电泳图；B：RcXYNC的编码序列；C：RcXYNC的预测保守结构域；D：RcXYNC与其他物种多序列比对分析；E：RcXYNC系统发育分析（“▲”处标记为RcXYNC所在位置）

Fig. 1 Bioinformatics characteristics of RcXYNC geneA: Electrophoresis map of gene coding sequence cloning. B: Coding sequence of RcXYNC. C: Predicted conservative structure domain of RcXYNC. D: Multiple sequences alignment of RcXYNCwith other species. E: Phylogenetic analysis of RcXYNC (The location of RcXYNC marked with a '▲')

图2 RcXYNC蛋白质二级结构、三级结构、信号肽和跨膜结构域预测A：RcXYNC蛋白质二级结构预测；B：RcXYNC蛋白质三级结构预测；C：RcXYNC信号肽预测；D：RcXYNC跨膜结构域预测

Fig. 2 Predicted secondary structure, tertiary structure, signal peptides and transmembrane domain of RcXYNCproteinA: Predicted secondary structure of RcXYNCprotein. B: Predicted tertiary structure of RcXYNCprotein. C: Predicted signal peptides of RcXYNC. D: Predicted transmembrane domain of RcXYNC

图3 RcXYNC蛋白的亚细胞定位GFP：绿色荧光蛋白；Chloroplast：叶绿体；Bright：明场；Merge：叠加场

Fig. 3 Subcellular localization of RcXYNC proteinGFP: Green fluorescent protein. Bright: Bright field. Merge: Integration of all fields

图4 蔷薇盘二孢侵染后RcXYNC的表达模式ns：无显著差异；*，P<0.05；**，P<0.01；***，P<0.001；****，P<0.000 1。下同

Fig. 4 Relative expression of RcXYNC in rose inoculated by H2O or M. rosaens: No significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.000 1. The same below

图5 RcXYNC的启动子序列及顺式作用元件分析A：基因启动子克隆凝胶电泳图；B：RcXYNC的启动子序列；C：RcXYNC启动子顺式作用元件示意图

Fig. 5 Analysis of RcXYNC promoter sequence and cis-acting elementsA: Electrophoresis map of gene promoter cloning. B: RcXYNC promoter sequence. C: Diagram of the cis-acting elements of the RcXYNC promoter

图6 瞬时过表达RcXYNC提高了月季对蔷薇盘二孢的敏感性A：瞬时过表达RcXYNC接种8 d后，R12-26叶盘的黑斑；B：瞬时过表达RcXYNC 接种5 d后，R12-26叶片中RcXYNC的相对表达量；C：瞬时过表达RcXYNC接种8 d后，R12-26叶盘的黑斑面积；D：瞬时过表达RcXYNC接种8 d后，R13-54叶盘的黑斑；E：瞬时过表达RcXYNC接种5 d后，R13-54叶片RcXYNC的相对表达量；F：瞬时过表达RcXYNC接种8 d后，R13-54叶盘的黑斑面积；G：瞬时过表达RcXYNC接种2 d后，R13-54和R12-26叶片的H2O2累积面积；H：瞬时过表达RcXYNC接种2 d后，R12-26和R13-54叶片的H2O2累积面积；I：瞬时过表达RcXYNC接种2 d后，R12-26叶片中抗性标志基因的表达量；J：瞬时过表达RcXYNC接种2 d后，R13-54叶片中抗性标志基因的表达量（WT：接种蔷薇盘二孢的月季叶片；PVX或PVX：：RcXYNC，被用携带PVX空载体或PVX：：RcXYNC重组载体的农杆菌悬浮液浸润后再接种蔷薇盘二孢的月季叶片）

Fig. 6 Transient overexpression of RcXYNC enhanced the sensitiveness of rose to M. rosaeA: The black spot phenotype of R12-26 infected with M. rosae under RcXYNC overexpression on 8 d. B: Relative expression of RcXYNC of R12-26 infected with M. rosae under RcXYNC overexpression on 5 d. C: The black spot area statistic of R12-26 infected with M. rosae under RcXYNC overexpression on 8 d. D: The black spot phenotype of R13-54 infected with M. rosae under RcXYNC overexpression on 8 d. E: Relative expression of RcXYNC of R13-54 infected with M. rosae under RcXYNC overexpression on 5 d. F: The black spot area statistic of R13-54 infected with M. rosae under RcXYNC overexpression on 8 d. G: The H2O2 accumulation area statistic of R12-26 and R13-54 infected with M. rosae under RcXYNC overexpression on 2 d. H: The H2O2 accumulation phenotype of R12-26 and R13-54 infected with M. rosae under RcXYNC overexpression on 2 d. I: Relative expression of defense genes in R12-26 infected with M. rosae under RcXYNC overexpression on 2 d. J: Relative expression of defense genes in R13-54 infected with M. rosae under RcXYNC overexpression on 2 d (WT: rose leaves inoculated with M. rosae; PVX or PVX::RcXYNC; rose leaves infiltrated with Agrobacterium suspensions carrying PVX empty vector or PVX::RcXYNC recombinant vector and subsequently inoculated with M. rosae)

图7 沉默RcXYNC增强了月季对蔷薇盘二孢的抗性A：瞬时沉默RcXYNC接种8 d后，R12-26叶盘上的黑斑；B：瞬时沉默RcXYNC接种5 d后，R12-26叶片RcXYNC的相对表达量；C：瞬时沉默RcXYNC接种8 d后，R12-26叶盘的黑斑面积；D：瞬时沉默RcXYNC接种8 d后，R13-54叶盘上的黑斑；E：瞬时沉默RcXYNC接种5 d后，R13-54叶片RcXYNC的相对表达量；F：瞬时沉默RcXYNC接种8 d后，R13-54叶盘上的黑斑面积；G：瞬时沉默RcXYNC接种2 d后，R13-54和R12-26叶片H2O2的累积面积；H：瞬时沉默RcXYNC 接种2 d后，R12-26和R13-54叶片H2O2的累积情况；I：瞬时沉默RcXYNC接种2 d后，R12-26叶片抗性标志基因的表达量；J：瞬时沉默RcXYNC接种2 d后，R13-54叶片抗性标志基因的表达量（WT：接种蔷薇盘二孢的月季叶片；TRV或TRV：：RcXYNC，被用携带TRV空载体或TRV：：RcXYNC重组载体的农杆菌悬浮液浸润后再接种蔷薇盘二孢的月季叶片）

Fig. 7 Silencing of RcXYNC enhanced the resistance of rose to M. rosaeA: The black spot phenotype of R12-26 infected with M. rosae under RcXYNC silencing on 8 d. B: Relative expression of RcXYNC of R12-26 infected with M. rosae under RcXYNC silencing on 5 d. C: The black spot area statistic of R12-26 infected with M. rosae under RcXYNC silencing on 8 d. D: The black spot phenotype of R13-54 infected with M. rosae under RcXYNC silencing on 8 d. E: Relative expression of RcXYNC of R13-54 infected with M. rosae under RcXYNC silencing on 5 d. F: The black spot area statistic of R13-54 infected with M. rosae under RcXYNC silencing on 8 d. G: The H2O2 accumulation area statistic of R12-26 and R13-54 infected with M. rosae under RcXYNC silencing on 2 d. H: The H2O2 accumulation phenotype of R12-26 and R13-54 infected with M. rosae under RcXYNC silencing on 2 d. I: Relative expression of defense genes in R12-26 infected with M. rosae under RcXYNC silencing on 2 d. J: Relative expression of defense genes in R13-54 infected with M. rosae under RcXYNC silencing on 2 d (WT: rose leaves inoculated with M. rosae; TRV or TRV::RcXYNC; rose leaves infiltrated with Agrobacterium suspensions carrying TRV empty vector or TRV::RcXYNC recombinant vector and subsequently inoculated with M. rosae)

图8 酵母双杂交验证RcXYNC与候选蛋白的互作关系A：诱饵载体自激活活性检测；B：候选蛋白的基因克隆（泳道M：DL2000 marker；其余泳道：候选蛋白基因CDS区域克隆片段）；C：酵母一对一验证RcXYNC与候选目标蛋白的互作关系（AD：pGADT7；BD：pGBKT7；AD-T+BD-Lam，AD+BD和AD+BD-RcXYNC：阴性对照；AD-T+BD-53：阳性对照；10-1、10-2、10-3、10-4：菌液的稀释梯度）

Fig. 8 Verifying the interaction between RcXYNC and candidate proteins via yeast two-hybridA: Self-activation identification of bait vector. B: Gene cloning of candidate proteins. (Lane M: DL2000 marker; other lanes: CDS cloning of candidate proteins) C: One-to-one validation of RcXYNC interaction with candidate target proteins in yeast (AD: pGADT7; BD: pGBKT7; AD-T+BD-Lam, AD+BD and AD+BD-RcXYNC: negative control; AD-T+BD-53: positive control; 10-1, 10-2, 10-3, 10-4: the dilution gradients of bacterial solution)

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