Cloning and Expression of SlCaM3,SlCaM4 and SlCaM5 Genes under Cold Stress in Tomato

doi:10.7668/hbnxb.20193489

Abstract

Abstract:

Calmodulin is an important Ca²⁺ receptor protein in plants,which plays an important role in calcium signaling pathway and stress resistance.It is of great significance to study the mechanism of CaM protein in tomato under low temperature stress for cold tolerance breeding of tomato.Tomato calmodulin genes SlCaM3, SlCaM4 and SlCaM5 were cloned from tomato varieties Heinz1706,LA3969,Jifen 2,Jifen 3 and Nongbofenba 15,and their sequences and protein sequences were analyzed by bioinformatics;the cis acting elements in the promoter regions of SlCaM3,SlCaM4 and SlCaM5 were analyzed by plantcare;Quantitative Real-time PCR was used to analyze the expression patterns of SlCaM3,SlCaM4 and SlCaM5 under 15,5 ℃ temperature stress in different tomato varieties,and the specific expression of SlCaM3,SlCaM4 and SlCaM5 in different tissues was analyzed in combination with RNA-seq data.The results showed that the coding sequence of SlCaM3,SlCaM4 and SlCaM5 were 450 bp,and their similarity was 93.63%;the encoded amino acid sequences were identical,belonging to acidic stable hydrophilic protein with typical conservative domain of cam protein.The analysis of cis acting elements showed that the promoter regions of the three genes contained not only the necessary core elements,but also a variety of biotic and abiotic stress response elements,and showed a complementary pattern.The analysis of the expression patterns of different degrees of low temperature stress showed that the expression patterns of SlCaM3,SlCaM4 and SlCaM5 in five tomato materials showed a trend of first decreasing and then increasing at 15 ℃,and the expression of SlCaM5 increased more significantly.At 5 ℃,SlCaM3 and SlCaM4 did not change significantly,but the expression level of SlCaM5 gene increased significantly in the later stage of treatment.The high expression of SlCaM5 gene in the process of tomato resistance to low temperature indicated that it maintained the translation level of cam protein under low temperature and ensured that the function of CaM protein was not affected.The analysis of SlCaM3,SlCaM4 and SlCaM5 specific expression in different tissues of Heinz1706 showed that SlCaM3 and SlCaM4 were highly expressed in meristem,while the expression of SlCaM5 different tissues was not significantly different.

Key words: Tomato, Calmodulin, Cold, Gene cloning, qRT-PCR, Expression analysis

WANG Peng, TIAN Zhejuan, ZHAO Xuefang, KANG Chen, WU Zhiming, LI Yadong, HUANG Jinan. Cloning and Expression of SlCaM3,SlCaM4 and SlCaM5 Genes under Cold Stress in Tomato[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 75-84. doi: 10.7668/hbnxb.20193489.

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Figures/Tables 7

References 31

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引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	产物大小/bp Product size	用途 Function
SlCaM3-F	CCCTCTCTCTCTTTCTTTGTAGAAAA	450	基因克隆
SlCaM3-R	AAAAAACAACTTCAGAAGAGGGAAA
SlCaM4-F	AAAAGCTCTGTGAATTAATCGATA	450	基因克隆
SlCaM4-R	AAGATTAAGATGAGTTGATCAATTC
SlCaM5-F	TTTTCTCTCATTTTCAACTTCC	450	基因克隆
SlCaM5-R	TAGGTAAGTTGAGTTGATCAGTTC
SlCaM3RT-F	TCCACAACTTTCTTCTTCTCCC	226	实时荧光定量PCR
SlCaM3RT-R	TACCATCAGCATCCACTTCATT
SlCaM4RT-F	CTGATGTGGATGGTGATGGAC	175	实时荧光定量PCR
SlCaM4RT-R	CATCCTAACCGGAAACAGAAAC
SlCaM5RT-F	TTCTTCTCATCTATTGTCCCATTTT	250	实时荧光定量PCR
SlCaM5RT-R	AACTCGGGGAAGTCAATGGT
Slβ-actin-F	CAGCACCTTCCAGCAGATGT	124	内参基因
Slβ-actin-R	AGAAGTCTTTTCCTTTCACCAAC

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	产物大小/bp Product size	用途 Function
SlCaM3-F	CCCTCTCTCTCTTTCTTTGTAGAAAA	450	基因克隆
SlCaM3-R	AAAAAACAACTTCAGAAGAGGGAAA
SlCaM4-F	AAAAGCTCTGTGAATTAATCGATA	450	基因克隆
SlCaM4-R	AAGATTAAGATGAGTTGATCAATTC
SlCaM5-F	TTTTCTCTCATTTTCAACTTCC	450	基因克隆
SlCaM5-R	TAGGTAAGTTGAGTTGATCAGTTC
SlCaM3RT-F	TCCACAACTTTCTTCTTCTCCC	226	实时荧光定量PCR
SlCaM3RT-R	TACCATCAGCATCCACTTCATT
SlCaM4RT-F	CTGATGTGGATGGTGATGGAC	175	实时荧光定量PCR
SlCaM4RT-R	CATCCTAACCGGAAACAGAAAC
SlCaM5RT-F	TTCTTCTCATCTATTGTCCCATTTT	250	实时荧光定量PCR
SlCaM5RT-R	AACTCGGGGAAGTCAATGGT
Slβ-actin-F	CAGCACCTTCCAGCAGATGT	124	内参基因
Slβ-actin-R	AGAAGTCTTTTCCTTTCACCAAC

顺式元件 Element	顺式作用元件数量 Number of cis-acting element			序列 Sequence	功能注释 Functional annotation
顺式元件 Element	SlCaM3	SlCaM4	SlCaM5	序列 Sequence	功能注释 Functional annotation
ABRE	1	5	2	ACGTG	参与脱落酸反应的顺式作用元件
ARE	5	2	1	AAACCA	无氧诱导必需的顺式作用调节元件
CAT-box	1	3	0	GCCACT	与分生组织表达相关的顺式作用调控元件
CGTCA-motif、	2	0	1	CGTCA/TGACG	参与茉莉酸甲酯反应的顺式作用调控元件
TGACG-motif
G-box	2	8	2	CACGAC/CACGTC/TAAACGTG/	参与光响应顺式作用调节元件
				TACGTG/CCACGTAA/TACGTG
GT1-motif	1	1	0	GGTTAA	光响应元件
LTR	1	1	0	CCGAAA	参与低温响应的顺式作用元件
MBS	1	1	1	CAACTG	参与干旱诱导能力的MYB结合位点
MRE	0	1	2	AACCTAA	参与光响应的MYB结合位点
P-box	0	1	0	CCTTTTG	赤霉素应答元件
TATC-box	1	0	0	TATCCCA	参与赤霉素应答顺式作用元件
TC-rich repeats	1	2	0	GTTTTCTTAC	参与防御和应激反应顺式作用元件
TCA-element	2	0	1	TCAGAAGAGG	参与水杨酸反应的顺式作用元件
TGA-element	2	0	0	AACGAC	生长素响应元件
CAAT-box	30	47	37	CAAAT/CAAT/CCAAT	启动子和增强子区域中常见顺式作用元件
TATA-box	39	40	37	TATA/ATATAT/TATAA/TATAAA/	转录起点-30左右的核心启动子元件
				TATAAAA/TATACA/TATATA/
				TATATAA/TATATAAATC

顺式元件 Element	顺式作用元件数量 Number of cis-acting element			序列 Sequence	功能注释 Functional annotation
顺式元件 Element	SlCaM3	SlCaM4	SlCaM5	序列 Sequence	功能注释 Functional annotation
ABRE	1	5	2	ACGTG	参与脱落酸反应的顺式作用元件
ARE	5	2	1	AAACCA	无氧诱导必需的顺式作用调节元件
CAT-box	1	3	0	GCCACT	与分生组织表达相关的顺式作用调控元件
CGTCA-motif、	2	0	1	CGTCA/TGACG	参与茉莉酸甲酯反应的顺式作用调控元件
TGACG-motif
G-box	2	8	2	CACGAC/CACGTC/TAAACGTG/	参与光响应顺式作用调节元件
				TACGTG/CCACGTAA/TACGTG
GT1-motif	1	1	0	GGTTAA	光响应元件
LTR	1	1	0	CCGAAA	参与低温响应的顺式作用元件
MBS	1	1	1	CAACTG	参与干旱诱导能力的MYB结合位点
MRE	0	1	2	AACCTAA	参与光响应的MYB结合位点
P-box	0	1	0	CCTTTTG	赤霉素应答元件
TATC-box	1	0	0	TATCCCA	参与赤霉素应答顺式作用元件
TC-rich repeats	1	2	0	GTTTTCTTAC	参与防御和应激反应顺式作用元件
TCA-element	2	0	1	TCAGAAGAGG	参与水杨酸反应的顺式作用元件
TGA-element	2	0	0	AACGAC	生长素响应元件
CAAT-box	30	47	37	CAAAT/CAAT/CCAAT	启动子和增强子区域中常见顺式作用元件
TATA-box	39	40	37	TATA/ATATAT/TATAA/TATAAA/	转录起点-30左右的核心启动子元件
				TATAAAA/TATACA/TATATA/
				TATATAA/TATATAAATC

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