Cold Resistance Function Analysis of Cassava MeMYC2.2

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

Abstract

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

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.

Figures/Tables 8

References 34

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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

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

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）

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）

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