木薯MeMYC2.2基因耐低温功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0461

摘要/Abstract

摘要：

MYC2（MYeloCytomatosis）转录因子是植物应对逆境胁迫过程中茉莉酸信号传导相关的核心转录因子。本研究旨在初步分析木薯MeMYC2.2基因在低温胁迫响应中的功能。利用生物信息学分析木薯MeMYC2.1和MeMYC2.2基因的结构及其编码蛋白的理化性质；通过定量PCR分析了上述2个基因在木薯组培苗叶片中对低温胁迫的响应；通过转基因拟南芥研究MeMYC2.2的抗冻功能。木薯组培苗叶片中2个MeMYC2基因的表达均在低温胁迫早期被诱导，其中，与MeMYC2.1相比，MeMYC2.2差异表达更显著。MeMYC2.2蛋白主要定位于细胞核中，且在酵母中具有明显转录自激活功能，表明该蛋白具有转录因子特性。与野生型相比，过表达MeMYC2.2的转基因拟南芥抗冻能力显著提高。在低温处理下，CBF3基因在转基因拟南芥中的表达量要明显高于其在野生型的表达量，但另外3个CBF基因在转基因拟南芥中的表达量明显下降。木薯MeMYC2.2的表达受低温和茉莉酸调控，可以提高植物的抗冻性，且可能影响CBF基因对低温的响应。本研究为进一步利用MeMYC2基因改良木薯的低温耐受性奠定了理论基础。

关键词: 木薯, MeMYC2, CBF通路, 低温胁迫

Abstract:

Jasmonic acid（JA）signal transduction is a key pathway in plant response to stresses, in which MYC2（MYeloCytomatosis）plays an important role. This study aims to investigate the function of MeMYC2.2 from cassava（Manihot esculenta Crantz）under cold stress. Bioinformatics was used to identify the structures and physicochemical properties of two MYC2 gene, namely MeMYC2.1 and MeMYC2.2 from cassava. Quantitative PCR was to analyze the responses of them in the leaves of tissue-cultured cassava seedling to cold stresses. Furthermore, the function of MeMYC2.2 for cold resistance was verified in transgenic Arabidopsis. As result, the expressions of two MeMYC2 genes were dramatically induced under early cold stress in the leaves of tissue-cultured cassava seedling, and the differential expression of MeMYC2.2 was more significant compared to MeMYC2.1. The MeMYC2.2 protein was predominantly localized in the nucleus and presented of transcriptional activation ability in yeast, suggesting this protein probably was characterized of a transcription factor. Over-expression of MeMYC2.2 resulted in significant improvement of freezing tolerance in transgenic Arabidopsis compared to wild one. Furthermore, the expression of CBF3 was significantly up-regulated by MeMYC2.2 transgenic in Arabidopsis under cold conditions. However, expressions of other three CBF genes were down-regulated by MeMYC2.2 transgenic Arabidopsis under cold conditions. The expression of MeMYC2.2 was regulated by cold and jasmonic acid, which may increase its cold tolerance and affect the responses of CBF genes to cold stress. This study lays a theoretical foundation in using MeMYC2.2 to improve cold resistance in cassava cultivars.

Key words: cassava, MeMYC2, CBF pathway, cold stress

于晓玲, 李文彬, 李智博, 阮孟斌. 木薯MeMYC2.2基因耐低温功能研究[J]. 生物技术通报, 2023, 39(1): 224-231.

YU Xiao-ling, LI Wen-bin, LI Zhi-bo, RUAN Meng-bin. Cold Resistance Function Analysis of Cassava MeMYC2.2[J]. Biotechnology Bulletin, 2023, 39(1): 224-231.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Primer usage
MeMYC 2.2-F	GCTCTAGAATGAATCTGT- GGACGGACGA	基因克隆 Gene cloning
MeMYC 2.2-R	GTGGATCCGGAGTCACCA- ACTTTGGTTG	基因克隆 Gene cloning
MeActin-qF	TGATGAGTCTGGTCCATCCA	内参基因
MeActin-qR	CCTCCTACGACCCAATCTCA	Reference gene
MeMYC 2.2-qF	CCCTGACCAGGGTGAGAATGAT	实时荧光定量 RT-qPCR
MeMYC 2.2-qR	CCCTTTCGACACATGCTGAT	实时荧光定量 RT-qPCR
AtActin1-qF	GGGCAAGTGATCACCATTGG	转基因拟南芥基因表达分析 Gene expression analysis of transgenic Arabidopsis
AtActin1-qR	TGGAGCCAAAGCAGTGATCTC
AtCBF1-qF	ATTATTGTCCGACGTTGGCCA
AtCBF1-qR	GCGAAGTTGAGACATGCTGAT
AtCBF2-qF	TGCCTGTCTCAATTTCGCTGA
AtCBF2-qR	CGGCCATGTTATCCAACAAAC
AtCBF3-qF	CAAGGATTTGGCTCGGAACAT
AtCBF3-qR	TCCACCAACGTCTCCTCCATG
AtCBF4-qF	AAGCTGCAATGGCGTTTCAGA
AtCBF4-qR	CGTCACCCACTCCGTCAAAGT

表2 MeMYC2生物信息学分析

Table 2 Bioinformatics analysis of MeMYC2

分析内容 Item	MeMYC2.1	MeMYC2.2
开放阅读框Open read frame（ORF）	2 055 bp	2 019 bp
内含子 Intron	-	-
蛋白质的分子量Molecular weight（Mw）	75 043.89	74 015.17
等电点 Isoelectric point（pI）	5.49	5.14
Plant-mPLoc软件预测 Plant-mPLoc software prediction	定位于细胞核	定位于细胞核
SignalP	非分泌蛋白	非分泌蛋白
保守功能结构域Conservative functional domains	bHLH-MYC保守结构域（N69-20），及HLH DNA结合域（N505-556）	bHLH-MYC保守结构域（N5-243），及HLH DNA结合域（N218-239）

图1 不同冷胁迫时间下MeMYC2的表达分析冷胁迫温度为10℃，样品为木薯种苗新生叶片；荧光定量PCR分析，内参基因为MeActin1；显著差异用不同字母表示（P≤0.05）；误差线为每组处理的标准误差（n=3），下同

Fig. 1 Expression patterns of MeMYC2 under different cold stress（CS）time In vitro cassava seedling leaves were treated by low temperature at 10℃. New expanding leaves of stressed or unstressed seedlings were used in qPCR analysis. The MeActin1 was used as housekeep gene. Error bars are ± SD（n=3）, different letters indicate significant differences with P≤0.05（ANOVA test）, the same below

图2 MeMYC2.2转录因子验证 A：亚细胞定位结果；B：酵母自激活实验

Fig. 2 Validation experiments of transcription factor MeMYC2.2 A：Subcellular localization results. B：Yeast autoactivation assay

图3 转基因拟南芥中MeMYC2.2表达分析

Fig. 3 Expression analysis of MeMYC2.2 in transgenic Arabidopsis plants

图4 转基因和野生型植株表现出不同的耐冻能力两周龄的幼苗置于-20℃低温处理4-6 h，然后在正常培养条件下恢复培养5 d

Fig. 4 Wild-type and transgenic plants demonstrating different freezing tolerance abilities Two-week-old seedlings were treated by cold at -20℃ for 4-6 h, and then these treated seedlings were recovered for 5 d under normal conditions

表3 野生型和转基因植株冷冻处理后的存活率统计

Table 3 Survival rates of wild-type and transgenic plants with cold treatment (n>30)

基因型 Genotype	冷冻处理 Freezing treatment/h	存活率 Survival rate/%
Col-0	4	100
	5	74.2±1.5
	6	29.4±7.1
MYC2.2 OE	4	100
	5	92.2±2.3
	6	83.6±3.6

图5 冷冻胁迫下转基因拟南芥中CBF基因的表达分析各CBF基因在未经处理的野生型拟南芥中的相对表达量设为1；显著差异用不同字母表示（P≤0.05）；误差线为每组处理的标准误差（n=3）

Fig. 5 Expression analysis of CBF genes in transgenic Arabidopsis under cold stress The relative expression of indicated CBF gene in Col-0 Arabidopsis under normal conditions was set to 1. Error bars are ± SD（n=3）, and different letters indicate differences with P≤0.05（ANOVA test）

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