苦荞FtCBL基因的鉴定及对干旱与高钙胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2021-1367

摘要/Abstract

摘要：

植物CBL基因在逆境胁迫应答方面具有重要作用,鉴定苦荞FtCBL基因,并探索其对干旱高钙胁迫的响应具有重要意义。利用生物信息学方法鉴定FtCBL基因,通过模拟胁迫实验,研究FtCBL基因在干旱、高钙和干旱+高钙下的表达规律。共鉴定到5个苦荞FtCBL基因,均具有EF-hand钙结合结构域,分别定位于Ft3（2个）、Ft4（1个）和Ft5（2个）染色体上,cDNA长度在642-684 bp间,编码蛋白含213-227个氨基酸,序列高度保守。干旱下苦荞CAT与APX活性急剧升高,但在高钙下差异不显著,说明苦荞较适宜高钙环境生长。干旱下FtCBL1在8 h、24 h下调表达,FtCBL2上调表达,FtCBL3-1在8 h上调表达;高钙下FtCBL1在8 h、24 h和FtCBL9在24 h下调表达,FtCBL2上调表达;干旱+高钙下FtCBL3-1在8 h、24 h上调表达。共鉴定到5个FtCBL基因,其中FtCBL1、FtCBL2响应干旱、高钙胁迫,FtCBL3-1响应干旱、干旱+高钙胁迫,FtCBL9响应高钙胁迫。

关键词: 苦荞, 类钙调磷酸酶B蛋白, 干旱胁迫, 高钙胁迫, 干旱高钙胁迫

Abstract:

Plant CBL genes play important roles in stress response. It is of great significance to identify FtCBL genes of tartary buckwheat（Fagopyrum tataricum L.）and to explore its responses to drought and high calcium stress. Bioinformatics methods were conducted to identify the FtCBL gene of tartary buckwheat. The expression of FtCBL gene under drought,high calcium and combined drought and high calcium treatment was investigated by mimicing stress. A total of 5 FtCBL genes with conserved EF-hand calcium-binding domain were identified,which were located at chromosome Ft3（2）,Ft4（1）and Ft5（2）. Their cDNA length was 642-684 bp,encoding 213-227 amino acids with highly conserved sequence. The CAT and APX enzyme activities of tartary buckwheat increased sharply under drought,but the difference was insignificant under high calcium,indicating that tartary buckwheat was relatively suitable for growing under high calcium environment. Under drought stress,FtCBL1 was down-regulated at 8 and 24 h,but FtCBL2 at 8 and 24 h and FtCBL3-1 at 8 h were up-regulated. Under high calcium stress,FtCBL1 at 8 and 24 h and FtCBL9 at 24 h were down-regulated,whereas FtCBL2 was up-regulated. Under combined drought and high calcium stress,FtCBL3-1 was up-regulated at 8 and 24 h. A total of 5 FtCBL genes are identified in tartary buckwheat. FtCBL1 and FtCBL2 are induced by drought stress and high calcium stress,respectively,FtCBL3-1 is induced by drought stress and combined drought and high calcium stress,and FtCBL9 is induced by high calcium stress.

Key words: Fagopyrum tataricum, calcineurin B-like protein, drought stress, high-calcium stress, drought and high-calcium stress

关志秀, 汪燕, 梁成刚, 韦春玉, 黄娟, 陈庆富. 苦荞FtCBL基因的鉴定及对干旱与高钙胁迫的响应[J]. 生物技术通报, 2022, 38(8): 101-109.

GUAN Zhi-xiu, WANG Yan, LIANG Cheng-gang, WEI Chun-yu, HUANG Juan, CHEN Qing-fu. Identification of FtCBL Genes in Fagopyrum tataricum and Their Stress Responses to Drought and High Calcium[J]. Biotechnology Bulletin, 2022, 38(8): 101-109.

图/表 9

表1 RT-qPCR基因表达的引物序列

Table 1 Primer sequences for RT-qPCR of gene expression

基因编号Gene No.	引物序列 Primer sequence（5'-3'）
FtPinG0000638500.01	F	CTGTGTCTCTTGCCTCCC	R	CAATGGCTCCCTTGTGCT
FtPinG0003042400.01	F	ACTTTCTGTGTTCCATCC	R	TACCACCATTTGCTTTAC
FtPinG0003981900.01	F	GGCACGAGGTCAAGCAAATG	R	TTGTCGCCATTCGGATCTGT
FtPinG0007340500.01	F	AGCATTTCCTCGCTTCGT	R	CATCGTCAATCACCGCAC
FtPinG0008915000.01	F	CTTTCTGTTTTCCATCCG	R	TCGTCCTTGTCAATCCTT
FtActin	F	gaaattcgcaagtaccagaagag	R	ccaacaaggtatgcctcagc

表2 苦荞FtCBL基因理化性质分析

Table 2 Physicochemical properties analysis of FtCBL in F. tataricum

基因编号 Gene No.	序列长度Sequence length/bp	氨基酸Amino acid/aa	分子量Molecular weight/Da	等电点pI	脂肪族氨基酸指数 Aliphatic index	染色体 Chromosome	α-螺旋 Alpha helix/%	延长链 Extended strand/%	β-折叠 Beta turn/%	无规卷曲 Random coil/%
FtPinG0003981900.01	642	213	24 343.7	4.8	86.01	Ft3	49.30	7.51	7.51	35.68
FtPinG0007340500.01	684	227	26 265.9	4.9	90.62	Ft3	54.63	6.17	8.37	30.84
FtPinG0003042400.01	678	225	25 755.3	4.8	89.69	Ft5	56.00	4.44	7.11	32.44
FtPinG0008915000.01	681	226	25 887.4	4.8	90.13	Ft4	53.98	7.08	7.08	31.86
FtPinG0000638500.01	642	213	24 305.5	4.7	86.95	Ft5	51.17	6.57	6.10	36.15

图1 苦荞FtCBL的染色体定位图

Fig.1 Chromosome location of FtCBL in F. tataricum

图2 苦荞FtCBL基因结构分析 A：FtCBL DNA结构分析;B：FtCBL启动子顺式元件分析

Fig.2 Analysis of FtCBL gene structure in F. tataricum A：Analysis of FtCBL DNA structure. B：Cis-element analysis of FtCBL promoter

图3 苦荞CBL家族蛋白序列的聚类图

Fig.3 Dendrogram of CBL family protein sequence in F. tataricum

图4 苦荞与拟南芥CBL家族蛋白序列的多重比较分析

Fig.4 Multiple comparison analysis of CBL family protein sequence in F. tataricum and Arabidopsis

图5 苦荞FtCBL蛋白结构域（A）与三维结构（B）预测 A中红色区域表示EF-hand-8结构,绿色区域表示EF-hand-7结构

Fig.5 Prediction of domain（A）and tertiary structure（B）in FtCBL protein in F. tataricum The red and green areas refer to the EF-hand-8 and EF-hand-7 structure,respectively in A

图6 不同胁迫对苦荞CAT与APX活性的影响 *与**分别表示差异处理与对照间数据差异达到显著与极显著水平,下同

Fig.6 Effects of different stress on the CAT and APX activities in F. tataricum * and ** refer to significant and extremely significant difference between treatment and control,respectively. The same below

图7 不同胁迫下苦荞FtCBL基因的表达分析

Fig. 7 Analysis of FtCBL expression in F. tataricum under different stress

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