山葡萄转录因子VaMYB4a互作蛋白的筛选与鉴定

doi:10.16420/j.issn.0513-353x.2021-1219

摘要/Abstract

摘要：

以抗寒种质山葡萄（Vitis amurensis Rupr. ）‘左山-1’叶片组织为试材，通过Gateway技术构建酵母均一化cDNA文库，库容量为1.36 × 10⁷ cfu ∙ mL^-1，外源基因插入片段均大于1 kb，重组率为100%，符合后续试验要求。以VaMYB4a^113-252 C端调控区段为诱饵，经酵母双杂交筛选获得17个候选互作蛋白，选取2个与非生物胁迫应答密切相关的候选互作蛋白分别在酵母、拟南芥原生质体中进行互作验证，发现锌指类转录因子VaCOL4、VaCOL5均与VaMYB4a全长及VaMYB4a^113-252存在互作关系。经系统进化与蛋白保守结构域分析，推测葡萄COL基因家族中VaCOL2与VaMYB4a具有潜在互作关系，进而通过BiFC验证表明，VaCOL2与VaMYB4发生蛋白互作；顺式作用元件预测表明VvCOL2、VvCOL4与VvCOL5启动子区包含多个激素及逆境应答相关元件，其中VvCOL2包括多个低温响应元件（LTRE）；低温胁迫下，VaMYB4a、VaCOL4与VaCOL5具有相同的表达模式，但VaCOL2表达模式不同。综上，VaMYB4a可与VaCOL2、VaCOL4及VaCOL5互作形成蛋白复合体以协同或拮抗的方式调控植物低温胁迫应答。

关键词: 山葡萄, 低温胁迫, Gateway酵母双杂交文库, VaMYB4a, VaCOL, 互作分析

Abstract:

In this study，the yeast homogenized cDNA library was constructed by Gateway technology using the leaf tissue of cold-tolerant Vitis amurensis Rupr.‘Zuoshan-1’as the test material，and the library capacity was 1.36 × 10⁷ cfu ∙ mL^-1. Using VaMYB4a^113-252 C-terminal regulatory region as bait，17 candidate interactive proteins were obtained by yeast two-hybrid screening. Two candidate proteins encoding zinc finger-like transcription factors，VaCOL4 and VaCOL5，closely related to abiotic stress response，were selected for interactive validation in yeast and Arabidopsis protoplasts，respectively. Bothcould interact with VaMYB4a and VaMYB4a1^113-252，respectively. The phylogenetic and protein conserved domain analyses suggested a potential interaction between VaCOL2 and VaMYB4a，which was further verified by BiFC to confirm their interaction. cis-regulatory acting element prediction showed that the promoter regions of VvCOL2，VvCOL4 and VvCOL5 contain multiple hormone and stress-response related elements，among which VvCOL2 included multiple low temperature response elements（LTRs）. VaMYB4a，VaCOL4 and VaCOL5 share the same expression pattern，but VaCOL2 is expressed differently under cold stress. Overall，VaMYB4a can interact with VaCOL2，VaCOL4，and VaCOL5 to form a protein complex to regulate the plant response to cold stress in a synergistic or antagonistic manner.

Key words: Vitis amurensis, cold stress, Gateway Y2H cDNA library, VaMYB4a, VaCOL, interactive analysis

中图分类号:

S633.1

俞沁含, 李俊铎, 崔莹, 王佳慧, 郑巧玲, 徐伟荣. 山葡萄转录因子VaMYB4a互作蛋白的筛选与鉴定[J]. 园艺学报, 2023, 50(3): 508-522.

YU Qinhan, LI Junduo, CUI Ying, WANG Jiahui, ZHENG Qiaoling, XU Weirong. Screening and Identification of Interacting Protein of VaMYB4a from Vitis amurensis[J]. Acta Horticulturae Sinica, 2023, 50(3): 508-522.

图/表 10

参考文献 51

[1]	Agarwal M, Hao Y J, Kapoor A, Dong C H, Fujii H, Zheng X, Zhu J K. 2006. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. Journal of Biological Chemistry, 281 (49):37635-37645.
[2]	An J P, An X H, Yao J F, Wang X N, You C X, Wang X F, Hao Y J. 2018a. BTB protein MdBT 2 inhibits anthocyanin and proanthocyanidin biosynthesis by triggering MdMYB9 degradation in apple. Tree Physiology, 38 (10):1578-1587. doi: 10.1093/treephys/tpy063 URL
[3]	An J P, Li R, Qu F J, You C X, Wang X F, Hao Y J. 2018b. R2R3-MYB transcription factor MdMYB 23 is involved in the cold tolerance and proanthocyanidin accumulation in apple. The Plant Journal, 96 (3):562-577. doi: 10.1111/tpj.14050 URL
[4]	An J P, Wang X F, Espley R V, Wang K L, Bi S Q, You C X, Hao Y J. 2020a. An apple B-box protein MdBBX37 modulates anthocyanin biosynthesis and hypocotyl elongation synergistically with MdMYBs and MdHY5. Plant Cell Physiology, 61 (1):130-143. doi: 10.1093/pcp/pcz185 URL
[5]	An J P, Wang X F, Zhang X W, Xu H F, Bi S Q, You C X, Hao Y J. 2020b. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnology Journal, 18 (2):337-353. doi: 10.1111/pbi.v18.2 URL
[6]	An J P, Wang X F, Zhang X W, You C X, Hao Y J. 2021. Apple B-box protein BBX37 regulates jasmonic acid mediated cold tolerance through the JAZ-BBX37-ICE1-CBF pathway and undergoes MIEL1-mediated ubiquitination and degradation. New Phytologist, 229 (5):2709-2729.
[7]	Bai S, Tao R, Tang Y, Yin L, Ma Y, Ni J, Yan X, Yang Q, Wu Z, Zeng Y, Teng Y. 2019. BBX16,a B-box protein,positively regulates light-induced anthocyanin accumulation by activating MYB 10 in red pear. Plant Biotechnology Journal, 17 (10):1985-1997. doi: 10.1111/pbi.v17.10 URL
[8]	Cáceres-Mella A, Talaverano M I, Villalobos-González L, Ribalta-Pizarro C, Pastenes C. 2017. Controlled water deficit during ripening affects proanthocyanidin synthesis,concentration and composition in Cabernet Sauvignon grape skins. Plant Physiology Biochemistry, 117:34-41. doi: 10.1016/j.plaphy.2017.05.015 URL
[9]	Chen P, Zhi F, Li X, Shen W, Yan M, He J, Chana B, Fan T, Zhou S, Ma F. 2022. Zinc-finger protein MdBBX7/MdCOL9,a target of MdMIEL 1 E3 ligase,confers drought tolerance in apple. Plant Physiology, 188 (1):540-559. doi: 10.1093/plphys/kiab420 URL
[10]	Chen Rugang, Gong Zhenhui, Lu Minghui, Li Dawei, Huang Wei. 2010. Research advanced of the transcription factors networks related to plant adverse environmental stress. Journal of Agricultural Biotechnology, 18 (1):126-134. (in Chinese)
	陈儒钢, 巩振辉, 逯明辉, 李大伟, 黄炜. 2010. 植物抗逆反应中的转录因子网络研究进展. 农业生物技术学报, 18 (1):126-134.
[11]	Chen Y, Chen Z, Kang J, Kang D M, Gu H, Qin G. 2013. AtMYB14 regulates cold tolerance in Arabidopsis. Plant molecular Biology Report, 31 (1):87-97.
[12]	Cheng X F, Wang Z Y. 2005. Overexpression of COL9,a CONSTANS-LIKE gene,delays flowering by reducing expression of CO and FT in Arabidopsis thaliana. The Plant Journal, 43 (5):758-768. doi: 10.1111/tpj.2005.43.issue-5 URL
[13]	Cheo D L, Titus S A, Byrd D R, Hartley J L, Temple G F, Brasch M A. 2004. Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination:functional analysis of multi-segment expression clones. Genome Research, 14 (10b):2111-2120. doi: 10.1101/gr.2512204 URL
[14]	Cho L H, Yoon J, An G. 2017. The control of flowering time by environmental factors. The Plant Journal, 90 (4):708-719. doi: 10.1111/tpj.2017.90.issue-4 URL
[15]	Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris N N, Walker A R, Robinson S P, Bogs J. 2009. The grapevine R2R3-MYB transcription factor VvMYBF 1 regulates flavonol synthesis in developing grape berries. Plant Physiology, 151 (3):1513-1530. doi: 10.1104/pp.109.142059 pmid: 19741049
[16]	Edery I, Chu L L, Sonenberg N, Pelletier J. 1995. An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture). Molecular Cellular Biology, 15 (6):3363-3371. doi: 10.1128/MCB.15.6.3363 URL
[17]	Gangappa S N, Botto J F. 2014. The BBX family of plant transcription factors. Trends in Plant Science, 19 (7):460-470. doi: 10.1016/j.tplants.2014.01.010 pmid: 24582145
[18]	Hou S, Zhao T, Yang D, Li Q, Liang L, Wang G, Ma Q. 2021. Selection and validation of reference genes for quantitative RT-PCR analysis in Corylus heterophylla Fisch. × Corylus avellana L. Plants (Basel), 10 (1):159.
[19]	Katzen F. 2007. Gateway recombinatorial cloning:a biological operating system. Expert Opinion on Drug Discovery, 2 (4):571-589. doi: 10.1517/17460441.2.4.571 URL
[20]	Landia M, Tattinib M, Gould K S. 2015. Multiple functional roles of anthocyanins in plant-environment interactions. Environmental Experimental Botany, 119:4 -17. doi: 10.1016/j.envexpbot.2015.05.012 URL
[21]	Lee H G, Seo P J. 2016. The Arabidopsis MIEL 1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96. Nature Communications, 7:12525. doi: 10.1038/ncomms12525
[22]	Li C, Pei J, Yan X, Cui X, Tsuruta M, Liu Y, Lian C. 2021a. A poplar B-box protein PtrBBX23 modulates the accumulation of anthocyanins and proanthocyanidins in response to high light. Plant Cell & Environment, 44 (9):3015-3033.
[23]	Li Y, Shi Y, Li M, Fu D, Wu S, Li J, Gong Z, Liu H, Yang S. 2021b. The CRY2-COP1-HY5-BBX7/ 8 module regulates blue light-dependent cold acclimation in Arabidopsis. The Plant Cell, 33 (11):3555-3573. doi: 10.1093/plcell/koab215 URL
[24]	Liu Y, Lin G, Yin C M, Fang Y. 2020. B-box transcription factor 28 regulates flowering by interacting with constans. Scientific Reports, 10 (1):17889. doi: 10.1038/s41598-020-74838-8
[25]	Lu Suwen, Zheng Xuanang, Wang Jiayang, Fang Jinggui. 2021. Research progress on the metabolism of flavonoids in grape. Acta Horticulturae Sinica, 48 (12):2506-2524. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0558 URL
	卢素文, 郑暄昂, 王佳洋, 房经贵. 2021. 葡萄类黄酮代谢研究进展. 园艺学报, 48 (12):2506-2524. doi: 10.16420/j.issn.0513-353x.2021-0558 URL
[26]	Meng L, Fan Z, Qiang Z, Wang C, Gao Ying, Deng Y, Zhu B, Zhu H, Chen J, Shan W, Yin X, Zhong S, Grierson D, Jiang C Z, Luo Y, Fu D-Q. 2018. BEL1-LIKE HOMEODOMAIN 11 regulates chloroplast development and chlorophyll synthesis in tomato fruit. The Plant Journal, 94 (6):1126-1140. doi: 10.1111/tpj.13924 pmid: 29659108
[27]	Merzlyak M N, Chivkunova O B. 2000. Light-stress-induced pigment changes and evidence for anthocyanin photoprotection in apples. Journal of Photochemistry Photobiology, 55 (2-3):155-163. doi: 10.1016/S1011-1344(00)00042-7 URL
[28]	Naing A H, Ai T N, Lim K B, Lee I J, Kim C K. 2018. Overexpression of Rosea 1 from snapdragon enhances anthocyanin accumulation and abiotic stress tolerance in transgenic tobacco. Frontiers in Plant Science, 9:1070. doi: 10.3389/fpls.2018.01070 pmid: 30158941
[29]	Ogata K, Morikawa S, Nakamura H, Hojo H, Yoshimura S, Zhang R, Aimoto S, Ametani Y, Hirata Z, Sarai A. 1995. Comparison of the free and DNA-complexed forms of the DMA-binding domain from c-Myb. Nature Structural Biology, 2 (4):309-320. doi: 10.1038/nsb0495-309 pmid: 7796266
[30]	Pei Lili, Guo Yuhua, Xu Zhaoshi, Li Liancheng, Chen Ming, Ma Youzhi. 2012. Research progress on stress-related protein kinases in plants. Acta Botanica Boreali-Occidentalia Sinica, 32 (5):1052-1061. (in Chinese)
	裴丽丽, 郭玉华, 徐兆师, 李连城, 陈明, 马有志. 2012. 植物逆境胁迫相关蛋白激酶的研究进展. 西北植物学报, 32 (5):1052-1061.
[31]	Peng Xiaoyu, Liu Rong, Yang Jing, Ding Xia, Wang Fang, Li Yangsheng, Cai Yaohui. 2014. Construction and identification of a mixed cDNA library of rice roots construction and identification of a mixed cDNA library of rice roots under salt and drought stress using Gateway technology. Chinese Journal of Applied and Environmental Biology,(2):291-294. (in Chinese)
	彭晓珏, 刘瑢, 阳菁, 丁霞, 王芳, 李阳生, 蔡耀辉. 2014. Gateway技术构建盐和干旱胁迫下水稻根系混合cDNA文库及其质量鉴定. 应用与环境生物学报,(2):291-294.
[32]	Piero A R L, Puglisi I, Rapisarda P, Petrone G. 2005. Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. Journal of Agricultural Food Chemistry, 53 (23):9083-9088. doi: 10.1021/jf051609s URL
[33]	Steinbach Y. 2019. The Arabidopsis thaliana CONSTANS-LIKE 4(COL4)- A modulator of flowering time. Frontiers in Plant Science, 28 (10):651.
[34]	Su L T, Li J W, Liu D Q, Zhai Y, Zhang H J, Li X W, Zhang Q L, Wang Y, Wang Q Y. 2014. A novel MYB transcription factor,GmMYBJ1,from soybean confers drought and cold tolerance in Arabidopsis thaliana. Gene, 538 (1):46-55. doi: 10.1016/j.gene.2014.01.024 URL
[35]	Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods. Molecular Biology Evolution, 28 (10):2731-2739. doi: 10.1093/molbev/msr121 URL
[36]	Thompson M A, Ramsay R G. 1995. Myb:an old oncoprotein with new roles. Bioessays, 17 (4):341-350. pmid: 7741726
[37]	Wang M, Hao J, Chen X, Zhang X. 2020. SlMYB 102 expression enhances low-temperature stress resistance in tomato plants. Peer J, 8:e10059. doi: 10.7717/peerj.10059 URL
[38]	Wang Shun-li, Xue Jing-qi, Zhu Fu-yong, Zhang Ping, Ren Xiu-xia, Liu Chuan-jiao,and Zhang Xiu-xin. 2014. Molecular cloning,expression and evolutionary analysis of the floweringregulating transcription factor gene PsCOL4 in tree peony. Acta Horticulturae Sinica, 41 (7):1409-1417. (in Chinese)
	王顺利, 薛璟祺, 朱富勇, 张萍, 任秀霞, 刘传娇, 张秀新. 2014. 牡丹开花调控转录因子基因PsCOL4的克隆、表达与系统进化分析. 园艺学报, 41 (7):1409-1417.
[39]	Wang X F, An J P, Liu X, Su L, You C X, Hao Y J. 2018. The nitrate-responsive protein MdBT 2 regulates anthocyanin biosynthesis by interacting with the MdMYB1 transcription factor. Plant Physiology, 178 (2):890-906. doi: 10.1104/pp.18.00244 URL
[40]	Wang Y, Mao Z, Jiang H, Zhang Z, Chen X. 2019. A feedback loop involving MdMYB108L and MdHY 5 controls apple cold tolerance. Biochemical Biophysical Research Communications, 512 (2):381-386. doi: 10.1016/j.bbrc.2019.03.101 URL
[41]	Xie Y P, Chen P, Yan Y, Bao C, Li X W, Wang L, Shen X, Li H, Liu X, Niu C, Zhu C, Fang N, Shao Y, Zhao T, Yu J T, Zhu J H, Xu L F, Nocker S V, Ma F, Guan Q M. 2018. An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple. New Phytologist, 218 (1):201-218. doi: 10.1111/nph.14952 URL
[42]	Xing C, Liu Y, Zhao L, Zhang S, Huang X. 2019. A novel MYB transcription factor regulates ascorbic acid synthesis and affects cold tolerance. Plant,Cell & Environment, 42 (3):832-845.
[43]	Yang A, Dai X, Zhang W H. 2012. A R2R3-type MYB gene,OsMYB2,is involved in salt,cold,and dehydration tolerance in rice. Journal of Experimental Botany, 63 (7):2541-2556. doi: 10.1093/jxb/err431 pmid: 22301384
[44]	Yang T W, He Y, Niu S B, Yan S W, Zhang Y. 2020. Identification and characterization of the CONSTANS(CO)/CONSTANS-like(COL)genes related to photoperiodic signaling and flowering in tomato. Plant Science, 301:110653. doi: 10.1016/j.plantsci.2020.110653 URL
[45]	Yao C, Li X, Li Y, Yang G, Liu W, Shao B, Zhong J, Huang P, Han D. 2022. Overexpression of a Malus baccata MYB transcription factor gene MbMYB4 increases cold and drought tolerance in Arabidopsis thaliana. International Journal of Molecular Sciences, 23 (3):1794. doi: 10.3390/ijms23031794 URL
[46]	Yao W, Wang L, Wang J, Ma F, Wang Y. 2017. VpPUB24,a novel gene from Chinese grapevine,Vitis pseudoreticulata,targets VpICE 1 to enhance cold tolerance. Journal of Experimental Botany, 68 (11):2933-2949. doi: 10.1093/jxb/erx136 URL
[47]	Yu Qin-han. 2022. Response mechanism of Amur grape VaMYB4a in response to cold stress[M. D. Dissertation]. Yinchuan: Ningxia University. (in Chinese)
	俞沁含. 2022. 山葡萄VaMYB4a参与低温胁迫应答的机理研究[硕士论文]. 银川: 宁夏大学.
[48]	Zhang H, Wang Z, Li X, Gao X, Dai Z, Cui Y, Zhi Y, Liu Q C, Zhai H, Gao S, Zhao N, He S Z. 2021. The IbBBX24-IbTOE3-IbPRX17 module enhances abiotic stress tolerance by scavenging reactive oxygen species in sweet potato. New Phytologist, 233 (3):1133-1152. doi: 10.1111/nph.17860 pmid: 34773641
[49]	Zhang L C, Zhao G, Xia C, Jia J Z, Liu X, Kong X Y. 2012. Overexpression of a wheat MYB transcription factor gene,TaMYB56-B,enhances tolerances to freezing and salt stresses in transgenic Arabidopsis. Gene, 505 (1):100-107. doi: 10.1016/j.gene.2012.05.033 URL
[50]	Zhang Y T, Liu Y, Hu W, Sun B, Chen Q, Tang H. 2018. Anthocyanin accumulation and related gene expression affected by low temperature during strawberry coloration. Acta Physiologiae Plantarum, 40 (11):1-8. doi: 10.1007/s11738-017-2577-4
[51]	Zhu J H, Verslues P E, Zheng X W, Lee B-h, Zhan X Q, Manabe Y, Sokolchik I, Zhu Y, Dong C H, Zhu J K, Hasegawa P M, Bressan R A. 2005. HOS10 encodes an R2R3-type MYB transcription factor essential for cold acclimation in plants. Proceedings of the National Academy of Sciences, 102 (28):9966-9971.

用途 Application	引物编号 Primer No.	引物序列（5′-3′） Primer sequence
构建载体	pDONR222-F	TCCCAGTCACGACGTTGTAAAACGACGGCCAGTCTT
Vector construction	pDONR222-R	AGAGCTGCCAGGAAACAGCTATGACCATGTAATACGACTC
	BD-VaMYB4a-F	atggccatggaggccgaattcATGGGGAGGTCTCCTTGCTG
	BD-VaMYB4a-R	ccgctgcaggtcgacggatccTTATTTCATCTCCAAACCTCTGTAATC
	BD-VaMYB4a^1-112-F	atggccatggaggccgaattcATGGGGAGGTCTCCTTGCTG
	BD-VaMYB4a^1-112-R	ctagttatgcggccgctgcagCTTTCTCCGTATGTGGGTGTTCC
	BD-VaMYB4a^113-252-F	atggccatggaggccgaattcAGCTTCTGAACCGAGGCATCG
	BD-VaMYB4a^113-252-R	ctagttatgcggccgctgcagTTATTTCATCTCCAAACCTCTGTAATC
	AD-VaCOL2-F	gccatggaggccagtgaattcATGTTGAAGGACGAAGGTTGTAACG
	AD-VaCOL2-R	cagctcgagctcgatggatccTTAGAATGACGGGACAATGCC
	AD-VaCOL4-F	gccatggaggccagtgaattcATGGTTGTTGAAGTGGAAAGTTGG
	AD-VaCOL4-R	cagctcgagctcgatggatccTCAAAACGATGGAACGACGC
	AD-VaCOL5-F	gccatggaggccagtgaattcATGGGATTCCAGGAACTTAACG
	AD-VaCOL5-R	cagctcgagctcgatggatccTTAATAGGACGGAACGACACCG
	pBI221CE-VaMYB4a-F	gagaacacgggggactctagaATGGATGCAGCTAAGGACGG
	pBI221CE-VaMYB4a-R	aaattcggtaccccgctcgagTTATTTCATCTCCAAACCTCTGTAATC
	pBI221CE-VaMYB4a^1-112-F	gagaacacgggggactctagaATGGATGCAGCTAAGGACGG
	pBI221CE-VaMYB4a^1-112-R	aaattcggtaccccgctcgagTCTCCGTATGTGGGTGTTCCA
	pBI221CE-VaMYB4a^113-252-F	gagaacacgggggactctagaAAGCTTCTGAACCGAGGCATC
	pBI221CE-MYB4a^113-252-R	aaattcggtaccccgctcgagTTATTTCATCTCCAAACCTCTGTAATC
	pBI221NE-COL2-F	cccaggcctactagtggatccATGTTGAAGGACGAAGGTTGTAACG
	pBI221NE-COL2-R	cccgggagcggtaccctcgagGAATGACGGGACAATGCC
	pBI221NE-COL4-F	cccaggcctactagtggatccATGGTTGTTGAAGTGGAAAGTTGG
	pBI221NE-COL4-R	cccgggagcggtaccctcgagTCAAAACGATGGAACGACGC
	pBI221NE-VaCOL5-F	cccaggcctactagtggatccATGGGATTCCAGGAACTTAACG
	pBI221NE-VaCOL5-R	cccgggagcggtaccctcgagATAGGACGGAACGACACCG
实时荧光定量PCR	VaMYB4a-RT-F	TCGCTTATATCCGGGCACAC
Quantitative Real-time PCR	VaMYB4a-RT-R	AGGTCAGGCCTCAGGTAGTT
	VaCOL2-RT-F	TTACAGCGTTCCGCACAAGA
	VaCOL2-RT-R	CCCCAGATGAAAACCCTGCT
	VaCOL4-RT-F	GGTGAACGACAACTGCTTTG
	VaCOL4-RT-R	GCACGACTCCCACTTCTAAA
	VvActin01-F	CTATCCTTCGTCTTGACCTTGCTG
	VvActin01-R	AGTGGTGAACATGTAACCCCTCTC
PCR检测	REC-F	GATGATGAAGATACCCCACCAAACCC
PCR detection	REC-R	GGTGCACGATGCACAGTTGAAGTG

用途 Application	引物编号 Primer No.	引物序列（5′-3′） Primer sequence
构建载体	pDONR222-F	TCCCAGTCACGACGTTGTAAAACGACGGCCAGTCTT
Vector construction	pDONR222-R	AGAGCTGCCAGGAAACAGCTATGACCATGTAATACGACTC
	BD-VaMYB4a-F	atggccatggaggccgaattcATGGGGAGGTCTCCTTGCTG
	BD-VaMYB4a-R	ccgctgcaggtcgacggatccTTATTTCATCTCCAAACCTCTGTAATC
	BD-VaMYB4a^1-112-F	atggccatggaggccgaattcATGGGGAGGTCTCCTTGCTG
	BD-VaMYB4a^1-112-R	ctagttatgcggccgctgcagCTTTCTCCGTATGTGGGTGTTCC
	BD-VaMYB4a^113-252-F	atggccatggaggccgaattcAGCTTCTGAACCGAGGCATCG
	BD-VaMYB4a^113-252-R	ctagttatgcggccgctgcagTTATTTCATCTCCAAACCTCTGTAATC
	AD-VaCOL2-F	gccatggaggccagtgaattcATGTTGAAGGACGAAGGTTGTAACG
	AD-VaCOL2-R	cagctcgagctcgatggatccTTAGAATGACGGGACAATGCC
	AD-VaCOL4-F	gccatggaggccagtgaattcATGGTTGTTGAAGTGGAAAGTTGG
	AD-VaCOL4-R	cagctcgagctcgatggatccTCAAAACGATGGAACGACGC
	AD-VaCOL5-F	gccatggaggccagtgaattcATGGGATTCCAGGAACTTAACG
	AD-VaCOL5-R	cagctcgagctcgatggatccTTAATAGGACGGAACGACACCG
	pBI221CE-VaMYB4a-F	gagaacacgggggactctagaATGGATGCAGCTAAGGACGG
	pBI221CE-VaMYB4a-R	aaattcggtaccccgctcgagTTATTTCATCTCCAAACCTCTGTAATC
	pBI221CE-VaMYB4a^1-112-F	gagaacacgggggactctagaATGGATGCAGCTAAGGACGG
	pBI221CE-VaMYB4a^1-112-R	aaattcggtaccccgctcgagTCTCCGTATGTGGGTGTTCCA
	pBI221CE-VaMYB4a^113-252-F	gagaacacgggggactctagaAAGCTTCTGAACCGAGGCATC
	pBI221CE-MYB4a^113-252-R	aaattcggtaccccgctcgagTTATTTCATCTCCAAACCTCTGTAATC
	pBI221NE-COL2-F	cccaggcctactagtggatccATGTTGAAGGACGAAGGTTGTAACG
	pBI221NE-COL2-R	cccgggagcggtaccctcgagGAATGACGGGACAATGCC
	pBI221NE-COL4-F	cccaggcctactagtggatccATGGTTGTTGAAGTGGAAAGTTGG
	pBI221NE-COL4-R	cccgggagcggtaccctcgagTCAAAACGATGGAACGACGC
	pBI221NE-VaCOL5-F	cccaggcctactagtggatccATGGGATTCCAGGAACTTAACG
	pBI221NE-VaCOL5-R	cccgggagcggtaccctcgagATAGGACGGAACGACACCG
实时荧光定量PCR	VaMYB4a-RT-F	TCGCTTATATCCGGGCACAC
Quantitative Real-time PCR	VaMYB4a-RT-R	AGGTCAGGCCTCAGGTAGTT
	VaCOL2-RT-F	TTACAGCGTTCCGCACAAGA
	VaCOL2-RT-R	CCCCAGATGAAAACCCTGCT
	VaCOL4-RT-F	GGTGAACGACAACTGCTTTG
	VaCOL4-RT-R	GCACGACTCCCACTTCTAAA
	VvActin01-F	CTATCCTTCGTCTTGACCTTGCTG
	VvActin01-R	AGTGGTGAACATGTAACCCCTCTC
PCR检测	REC-F	GATGATGAAGATACCCCACCAAACCC
PCR detection	REC-R	GGTGCACGATGCACAGTTGAAGTG

序号 Clone No.	NCBI比对结果 NCBI comparison results	E值 E-value	相似度/% Identity	登录号 Accession
VaMYB4a^113-252-1	dTDP-4-脱氢甘露糖还原酶 dTDP-4-dehydrorhamnose reductase	6.00E-103	64	XP_002282339.1
VaMYB4a^113-252-2	Nudix水解酶2 Nudix hydrolase 2	1.00E-160	100	RVW33646.1
VaMYB4a^113-252-3	抗锈蛋白激酶Lr10 Rust resistance kinase Lr10	9.00E-179	77	RVW52296.1
VaMYB4a^113-252-4	无活性的亮氨酸富集重复类受体蛋白激酶 PREDICTED：probably inactive leucine-rich repeat receptor-like protein kinase	0.0	82	XP_003635622.1
VaMYB4a^113-252-5	BEL1-like同源蛋白2 BEL1-like homeodomain protein 2	5e-61	100	XP_034683092.1
VaMYB4a^113-252-6	类几丁质酶蛋白2 Chitinase-like protein 2	1.00E-161	100	RVW69527.1
VaMYB4a^113-252-7	紫色酸性磷酸酶 PREDICTED：purple acid phosphatase 3	0.0	99	XP_002285160.1
VaMYB4a^113-252-8	锌指蛋白 CONSTANS-like 4 Zinc finger protein CONSTANS-like 4	9.00E-141	100	RVW58188.1
VaMYB4a^113-252-9	噻胺噻唑合成酶1，叶绿体 Thiamine thiazole synthase 1，chloroplastic	0.0	91	XP_002281769.1
VaMYB4a^113-252-10	半乳糖醛酸转移酶13亚型X1 Probable galacturonosyltransferase 13 isoform X1	0.0	99	XP_034708194.1
VaMYB4a^113-252-11	蛋白质S-酰基转移酶24 Protein S-acyltransferase 24	0.0	92	XP_002262878.1
VaMYB4a^113-252-12	ABC转运蛋白F家族成员1 ABC transporter F family member 1	6.00E-166	86	XP_010651804.1
VaMYB4a^113-252-13	萌发特异性半胱氨酸蛋白酶 Germination-specific cysteine protease 1	0.0	87	XP_010651804.1
VaMYB4a^113-252-14	过氧化氢酶同工酶1 PREDICTED：catalase isozyme 1	4.00E-168	86	XP_010663966.1
VaMYB4a^113-252-15	mRNA前体切割因子Im 25kDa亚基2亚型X1 PREDICTED：pre-mRNA cleavage factor Im 25 kDa subunit 2 isoform X1	2.00E-167	99	XP_002267257.1
VaMYB4a^113-252-16	GDP-L-半乳糖磷酸化酶1 GDP-L-galactose phosphorylase 1	3.00E-31	57	RVW30017.1
VaMYB4a^113-252-17	锌指蛋白CONSTANS-like 5 Zinc finger protein CONSTANS-like 5	1.00E-163	75	RVW79681.1

序号 Clone No.	NCBI比对结果 NCBI comparison results	E值 E-value	相似度/% Identity	登录号 Accession
VaMYB4a^113-252-1	dTDP-4-脱氢甘露糖还原酶 dTDP-4-dehydrorhamnose reductase	6.00E-103	64	XP_002282339.1
VaMYB4a^113-252-2	Nudix水解酶2 Nudix hydrolase 2	1.00E-160	100	RVW33646.1
VaMYB4a^113-252-3	抗锈蛋白激酶Lr10 Rust resistance kinase Lr10	9.00E-179	77	RVW52296.1
VaMYB4a^113-252-4	无活性的亮氨酸富集重复类受体蛋白激酶 PREDICTED：probably inactive leucine-rich repeat receptor-like protein kinase	0.0	82	XP_003635622.1
VaMYB4a^113-252-5	BEL1-like同源蛋白2 BEL1-like homeodomain protein 2	5e-61	100	XP_034683092.1
VaMYB4a^113-252-6	类几丁质酶蛋白2 Chitinase-like protein 2	1.00E-161	100	RVW69527.1
VaMYB4a^113-252-7	紫色酸性磷酸酶 PREDICTED：purple acid phosphatase 3	0.0	99	XP_002285160.1
VaMYB4a^113-252-8	锌指蛋白 CONSTANS-like 4 Zinc finger protein CONSTANS-like 4	9.00E-141	100	RVW58188.1
VaMYB4a^113-252-9	噻胺噻唑合成酶1，叶绿体 Thiamine thiazole synthase 1，chloroplastic	0.0	91	XP_002281769.1
VaMYB4a^113-252-10	半乳糖醛酸转移酶13亚型X1 Probable galacturonosyltransferase 13 isoform X1	0.0	99	XP_034708194.1
VaMYB4a^113-252-11	蛋白质S-酰基转移酶24 Protein S-acyltransferase 24	0.0	92	XP_002262878.1
VaMYB4a^113-252-12	ABC转运蛋白F家族成员1 ABC transporter F family member 1	6.00E-166	86	XP_010651804.1
VaMYB4a^113-252-13	萌发特异性半胱氨酸蛋白酶 Germination-specific cysteine protease 1	0.0	87	XP_010651804.1
VaMYB4a^113-252-14	过氧化氢酶同工酶1 PREDICTED：catalase isozyme 1	4.00E-168	86	XP_010663966.1
VaMYB4a^113-252-15	mRNA前体切割因子Im 25kDa亚基2亚型X1 PREDICTED：pre-mRNA cleavage factor Im 25 kDa subunit 2 isoform X1	2.00E-167	99	XP_002267257.1
VaMYB4a^113-252-16	GDP-L-半乳糖磷酸化酶1 GDP-L-galactose phosphorylase 1	3.00E-31	57	RVW30017.1
VaMYB4a^113-252-17	锌指蛋白CONSTANS-like 5 Zinc finger protein CONSTANS-like 5	1.00E-163	75	RVW79681.1