柑橘溃疡病抗性相关转录因子CitMYB20的功能

doi:10.3864/j.issn.0578-1752.2020.10.007

Abstract

Abstract:

【Objective】The objective of this study is to reveal the response of CitMYB20 to Xanthomonas citri subsp. citri (Xcc) and exogenous phytohormones in different citrus varieties, evaluate the resistance of transgenic plants to citrus bacterial canker (CBC), and to verify the biological function of CitMYB20.【Method】The primer pairs were designed according to the Citrus genome, the open reading frame sequences and promoter sequences of CitMYB20 were cloned from CBC resistant variety Fortunella japonica and CBC sensitive variety Poncirus trifoliata by PCR. The cis-acting elements of promoter sequences were predicted using online software PlantCARE. The detached mature leaves from F. japonica and P. trifoliata were inoculated with Xcc by acupuncture method, and collected at 0, 1, 3, and 5 d after inoculation. The citrus leaves were collected after being treated with exogenous salicylic acid, methyl jasmonate, and ethephon respectively for 0, 12, 24, 36, and 48 h. The expression of CitMYB20 was detected by qRT-PCR using the above treated leaves. The over-expressed vector and RNA interference vector of CitMYB20 were constructed through inserting the target sequences into the pLGNe plasmid. The transgenic plants were obtained through Agrobacterium tumefaciens, and the mature leaves of transgenic lines were randomly selected to conduct resistance evaluation against CBC.【Result】The open reading frames of CitMYB20 in F. japonica (GenBank number MN689609) and P. trifoliata (GenBank number MN689608) were highly conservative, with a similarity of 99%. There were abscisic acid-responsive and methyl jasmonate-responsive cis-acting elements in the promoter sequence of CitMYB20 from two varieties of citrus. However, there was a salicylic acid-responsive element in the promoter sequence from F. japonica (GenBank number MN689611), but not in the sequence from P. trifoliata (GenBank number MN689610). At the 5th day of Xcc in vitro inoculation, the expression of CitMYB20 in mature leaves of F. japonica was increased significantly, reaching 2.5 times of the healthy control, while the expression of CitMYB20 in P. trifoliata did not show significantly change during the whole infection period. During exogenous hormone treatment, the response of CitMYB20 to salicylic acid and methyl jasmonate was similar, that is, it showed the trend of first increasing and then decreasing in F. japonica, while the expression of CitMYB20 in P. trifoliata was decreased firstly and then returned to normal. When the leaves were treated with exogenous ethephon, the expression of CitMYB20 in F. japonica was decreased slightly, while in P. trifoliata, it showed the trend of first increasing and then decreasing. A total of 7 over-expressed plants and 5 RNA-interfered ones were obtained through genetic transformation. There was no significant difference in morphological development between transgenic plants and control plants. The results of disease resistance evaluation showed that the CitMYB20 over-expressed plants could reduce the symptom of CBC, while the RNA-interfered plants were more sensitive to Xcc.【Conclusion】The expression of CitMYB20 was induced by Xcc in CBC resistant variety. CitMYB20 is a positive regulatory transcription factor against Xcc, its function against CBC may require salicylic acid and jasmonic acid signaling pathway. CitMYB20 can be used as a candidate gene for genetically modified breeding against CBC.

Key words: Xanthomonas citri subsp. citri (Xcc), citrus bacterial canker (CBC), R2R3-MYB transcription factor, overexpression, RNA interference (RNAi), disease resistance

YAO LiXiao,FAN HaiFang,ZHANG QingWen,HE YongRui,XU LanZhen,LEI TianGang,PENG AiHong,LI Qiang,ZOU XiuPing,CHEN ShanChun. Function of Citrus Bacterial Canker Resistance-Related Transcription Factor CitMYB20[J].Scientia Agricultura Sinica, 2020, 53(10): 1997-2008.

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引物名称 Primer name	用途 Amplification	引物序列 Primer sequence (5′-3′)	备注 Remark
CitMYB20-F	克隆基因开放阅读框 To clone ORF sequence	ATAGGATCCATGGGGAGGGCTCCCTG	BamH I酶切位点 BamH I restriction site
CitMYB20-R	克隆基因开放阅读框 To clone ORF sequence	CCGGAATTCCTAAAATGGTAATGTTAATGAGTCTGC	EcoR I酶切位点 EcoR I restriction site
CitMYB20-F (g)	克隆基因抑制表达片段 To clone RNAi fragment sequence	GCTCTAGAGGCGCGCCAAGCAAAACCAGAAGGCC	Xba I和Asc I酶切位点 Xba I and Asc I restriction site
CitMYB20-R (g)	克隆基因抑制表达片段 To clone RNAi fragment sequence	CGCGGATCCATTTAAATACCGATCGCAAATTCATTAAG	BamH I和Sma I酶切位点 BamH I and Sma I restriction site
CitMYB20-F (P)	克隆启动子 To clone promoter sequence	CCAGAACCTTTACTTTAATTTCTATTTTTA
CitMYB20-R (P)	克隆启动子 To clone promoter sequence	CTTGTTAATTTCTTTCAAAGTAGTGGAG
CitMYB20-F (q)	检测CitMYB20的表达量 To detect CitMYB20 expression	CTCCTCGGTCACTACTGGAGA
CitMYB20-R (q)	检测CitMYB20的表达量 To detect CitMYB20 expression	CATTAAGGCCGCCTCCGAAA
actin-f	检测actin的表达量 To detect actin expression	CATCCCTCAGCACCTTCC
actin-r	检测actin的表达量 To detect actin expression	CCAACCTTAGCACTTCTCC
35S-F	鉴定过表达植株 To identify over-expressed plant	GGAGTCAAAGATTCAAATAGAGGACCTAAC
CitMYB20-R (OE)	鉴定过表达植株 To identify over-expressed plant	TGACCACCTATTCCCCAACATGT
CitMYB20-F (RNAi)	鉴定RNAi抑制表达植株 To identify RNAi plant	ATTTGCGATCGGTATTTAAATGTGTAA
CitMYB20-R (RNAi)	鉴定RNAi抑制表达植株 To identify RNAi plant	GCTAGCCAGGATCCAAATACCTGCAAA

元件名称 Motif name	序列 Sequence	位置Position		方向Strand		功能 Function
元件名称 Motif name	序列 Sequence	枣阳小叶枳 P. trifoliata	金弹金柑 F. japonica	枣阳小叶枳 P. trifoliata	金弹金柑 F. japonica	功能 Function
ABRE	ACGTG	-1198	-1190	+	+	脱落酸响应 Abscisic acid responsiveness
CGTCA-motif	CGTCA	-321	-321	-	-	茉莉酸甲酯响应 Methyl jasmonate responsiveness
		-316		+
TGACG-motif	TGACG	-321	-321	+	+
		-316		-
TCA-element	CCATCTTTTT		-1452		-	水杨酸响应 Salicylic acid responsiveness
TCA-element	CCATCTTTTT		-1390		+	水杨酸响应 Salicylic acid responsiveness

Function of Citrus Bacterial Canker Resistance-Related Transcription Factor CitMYB20

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