叶绿素结合蛋白CP24介导光照响应基因StRSM 1调控叶绿素积累

doi:10.13560/j.cnki.biotech.bull.1985.2020-0502

摘要/Abstract

摘要：

单MYB转录因子成员RSM（RADIALIS-like SANT/MYB）突变,倒置黑暗诱导的叶绿素减少,原因有待确定;为了揭示RSM如何调控叶绿素积累,本研究运用基因工程途径,获得了普通烟草和马铃薯体内抑制和过量表达StRSM 1阳性转化株系,测定了阳性转化株系的叶绿素积累等生理表型;结果显示,普通烟草和马铃薯体内抑制RSM 1表达,显著增加了叶绿素积累,叶色随之加深;RSM 1过量表达,显著减少叶绿素积累,叶色变浅。叶绿素代谢相关基因表达测定结果显示,StRSM 1过量表达增加了黑暗下叶绿素结合蛋白CP24基因的表达,改变了其表达模式。以上结果表明,转录因子StRSM 1响应光照反向调控叶绿素积累,叶绿素结合蛋白CP24参与了StRSM 1对叶绿素积累的调控。结果有助于进一步明确RSM 1如何响应光照和深刻理解RSM 1参与的光照响应。

关键词: StRSM 1, 叶绿素结合蛋白, 叶绿素

Abstract:

Mutation on RSM（RADIALIS-like SANT/MYB）,a member of the single MYB transcription factor family inverts darkness-induced chlorophyll reduction,and the reason for it is unknown yet. To reveal how RSM regulates chlorophyll accumulation,StRSM 1-overexpressed positive transformant line of Nicotiana tabacum and Solanum tuberosum were generated,and chlorophyll accumulation and other physiologic phenotypes were assayed. The result showed that over-expression of RSM1 led to light green leaf and a less chlorophyll accumulation,and inhibition of RSM 1 expression increased chlorophyll accumulation. Expression assay of genes encoding chlorophyll metabolism and binding demonstrated that over-expression of StRSM 1 increased the expression of chlorophyll-binding protein CP24 under darkness and changed expression pattern of CP24. The above findings indicate that light-responding transcription factor StRSM 1 negatively regulates chlorophyll accumulation. CP24 involves in the regulation. The result will play an important role in further clarifying how RSM 1responds to light and deeply understanding the light response involved in RSM1.

Key words: StRSM 1, chlorophyll-binding protein, chlorophyll

撒世娟, 伍涵宇, 张晓萍, 郑蕊, 姚新灵. 叶绿素结合蛋白CP24介导光照响应基因StRSM 1调控叶绿素积累[J]. 生物技术通报, 2021, 37(1): 198-204.

SA Shi-juan, WU Han-yu, ZHANG Xiao-ping, ZHENG Rui, YAO Xin-ling. Light-responding Gene StRSM 1 Mediated by Chlorophyll-binding Protein CP24 Regulates Chlorophyll Accumulation[J]. Biotechnology Bulletin, 2021, 37(1): 198-204.

图/表 5

表1 引物序列

引物名称	序列（5'-3'）	用途
RSMPF	acgagctcggtaccatgagttcgatg	StRSM 1体内过量表达载体构建
RSMPR	acggtaccgagctccttagtcagagg	StRSM 1体内过量表达载体构建
RSMMF	acggtaccggtaccatgagttcgatg	StRSM 1体内抑制表达载体构建
RSMMR	acgagctcgagctccttagtcagagg	StRSM 1体内抑制表达载体构建
qRSMF	acacggttcttcaggatcatgg	StRSM 1 mRNA积累定量测定
qRSMR	ttcttcagcagtcttgcctcca	StRSM 1 mRNA积累定量测定
SRSMF	cacggttcttcaggatcatg	重组载体阳性鉴定
SRSMR	ggggaagggcaccatac	重组载体阳性鉴定
L25F	cccctcaccacagagtctgc	StRSM 1体内表达定量测定内参
L25R	aagggtgttgttgtcctcaatctt	StRSM 1体内表达定量测定内参
612F	agaggaaaaatgcccaatatc	CP 24 mRNA积累的定性测定
612R	gcttatggggtttgtttgtg	CP 24 mRNA积累的定性测定

图1 载体pC-StRSM 1+和pC-StRSM 1-构建过程示意图

图2 StRSM 1与不同同源物氨基酸序列比对 A：4个StRSMs与马铃薯RSM1比对;B：StRSMs与拟南芥比对;C：茄科不同植物RSMs比对

图3 ntrsm和strsm株系RSM1 mRNA和叶绿素积累测定结果 A：ntrsm株系RSM 1 mRNA积累;B：strsm株系RSM 1 mRNA积累;C：ntrsm和strsm株系表型;D：strsm株系叶绿素积累;E：ntrsm叶绿素积累

图4 随RSM 1过量表达的CP24基因RT-PCR产物 A：过量表达RSM 1普通烟草CP24基因mRNA积累;B：过量表达RSM 1马铃薯CP24基因mRNA积累3 讨论

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