草业学报 ›› 2023, Vol. 32 ›› Issue (3): 107-117.DOI: 10.11686/cyxb2022104
• 研究论文 • 上一篇
收稿日期:
2022-03-02
修回日期:
2022-05-18
出版日期:
2023-03-20
发布日期:
2022-12-30
通讯作者:
李淑霞
作者简介:
E-mail: lishuxia620@163.com基金资助:
Yuan WANG(), Jing WANG, Shu-xia LI()
Received:
2022-03-02
Revised:
2022-05-18
Online:
2023-03-20
Published:
2022-12-30
Contact:
Shu-xia LI
摘要:
盐胁迫是影响植物生长发育的主要非生物胁迫因子之一,BBX家族转录因子在植物色素积累、光形态发生、种子生长发育以及逆境适应等方面具有重要的调节作用。为明确紫花苜蓿BBX基因的功能,本研究使用Primer Premier 5软件根据NCBI数据库MsBBX24基因的序列设计特异性引物,以紫花苜蓿的cDNA为模板克隆MsBBX24基因。利用生物信息学软件对基因序列和结构进行分析,并与其他植物的BBX24进行比对和进化树构建,分析它们之间的进化关系。采用实时荧光定量PCR(qRT-PCR)分析MsBBX24基因的表达模式。利用DNA的酶切与连接方法构建MsBBX24过表达载体,采用农杆菌介导法将其转入野生型拟南芥,通过除草剂Basta筛选,以半定量RT-PCR鉴定获得阳性转基因植株。通过对野生型和MsBBX24转基因拟南芥植株的表型观察和生理指标测定分析它们的耐盐性。研究结果表明,MsBBX24基因编码区序列长723 bp,编码240个氨基酸,分子量为30.58 kDa,等电点为7.74,MsBBX24与鹰嘴豆和蒺藜苜蓿的BBX24具有较高的同源性。qRT-PCR检测结果表明,MsBBX24基因的表达量受盐胁迫(150 mmol·L-1 NaCl)诱导。在拟南芥中过表达MsBBX24基因分析表明,在NaCl胁迫下,MsBBX24基因的过表达能够促进幼苗根和侧根的生长,提高子叶绿化率,同时显著降低了转基因植株的相对电导率(IL)、丙二醛(MDA)和过氧化氢(H2O2)含量,提高了叶绿素(Chl)和脯氨酸(Pro)含量、过氧化氢酶(POD)和超氧化物歧化酶(SOD)活性。MsBBX24基因响应盐胁迫,主要通过提高抗氧化防御系统清除活性氧以增强转基因植株的耐盐性,MsBBX24基因可为紫花苜蓿耐盐分子育种提供重要的候选基因。
王园, 王晶, 李淑霞. 紫花苜蓿MsBBX24基因的克隆及耐盐性分析[J]. 草业学报, 2023, 32(3): 107-117.
Yuan WANG, Jing WANG, Shu-xia LI. Cloning of MsBBX24 from alfalfa (Medicago sativa) and determination of its role in salt tolerance[J]. Acta Prataculturae Sinica, 2023, 32(3): 107-117.
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 用途 Application |
---|---|---|
MsBBX24-F | ATGAAAATACAGTGTGATGT | 基因克隆 Gene cloning |
MsBBX24-R | TTAGCCGAAATCTGGCACCA | 基因克隆 Gene cloning |
cMsBBX24-F | TCGAGCTCAAGCTTCGAATTCATGAAAATACAGTGTGATGTGTG | 载体构建 Vector construction |
cMsBBX24-R | GTACCGTCGACTGCAGAATTCGCCGAAATCTGGCACC | 载体构建 Vector construction |
qMsBBX24-F | GCATCCTCTTGGGCTGTTGA | 荧光定量Real-time quatitative PCR (qRT-PCR) |
qMsBBX24-R | TCTGGCACCATGAAGTGCTC | 荧光定量Real-time quatitative PCR (qRT-PCR) |
MsActin-F | TTTGAGACTTTCAATGTGCCCGCC | 内参基因 Reference gene |
MsActin-R | TAGCATGTGGGAGTGCATAACCCT | 内参基因 Reference gene |
AtActin-F | GCCATCCAAGCTGTTCTCTC | 内参基因 Reference gene |
AtActin-R | GCTCGTAGTCAACAGCAACAA | 内参基因 Reference gene |
表1 本试验所用引物
Table 1 Primers used in this experiment
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) | 用途 Application |
---|---|---|
MsBBX24-F | ATGAAAATACAGTGTGATGT | 基因克隆 Gene cloning |
MsBBX24-R | TTAGCCGAAATCTGGCACCA | 基因克隆 Gene cloning |
cMsBBX24-F | TCGAGCTCAAGCTTCGAATTCATGAAAATACAGTGTGATGTGTG | 载体构建 Vector construction |
cMsBBX24-R | GTACCGTCGACTGCAGAATTCGCCGAAATCTGGCACC | 载体构建 Vector construction |
qMsBBX24-F | GCATCCTCTTGGGCTGTTGA | 荧光定量Real-time quatitative PCR (qRT-PCR) |
qMsBBX24-R | TCTGGCACCATGAAGTGCTC | 荧光定量Real-time quatitative PCR (qRT-PCR) |
MsActin-F | TTTGAGACTTTCAATGTGCCCGCC | 内参基因 Reference gene |
MsActin-R | TAGCATGTGGGAGTGCATAACCCT | 内参基因 Reference gene |
AtActin-F | GCCATCCAAGCTGTTCTCTC | 内参基因 Reference gene |
AtActin-R | GCTCGTAGTCAACAGCAACAA | 内参基因 Reference gene |
图1 MsBBX24基因的核苷酸序列及其编码的氨基酸序列ATG:起始密码子;*:终止密码子;横线标记:保守区域。ATG: Initiation codon; *: Temination codon; Horizontal line mark: Conserved domains.
Fig.1 Nucleotide and predicted amino acid sequences of MsBBX24 gene
图2 MsBBX24蛋白生物信息学分析A:MsBBX24蛋白质亲水性分析;B:MsBBX24蛋白跨膜结构域预测;C:MsBBX24蛋白二级结构预测(图中线条由高到低依次表示:α-螺旋、延伸链、β-转角、不规则卷曲)。A: Hydrophilicity analysis of MsBBX24 protein; B: Transmembrane structure prediction of MsBBX24 protein; C: Secondary structure prediction of MsBBX24 protein (The lines in the figure were alpha helix, extended strand, beta turn and random coil in descending order).
Fig.2 Bioinformatics analysis of MsBBX24 protein
图5 MsBBX24基因的表达量分析A:组织特异性表达,**表示根和叶表达量差异极显著(P<0.01);B:150 mmol·L-1 NaCl处理下根和叶的表达量,*和**分别表示同一组织中不同处理时间的表达量与0 h相比差异显著(P<0.05)和差异极显著(P<0.01)。A: Tissue-specific expression, ** indicated that significant differences in root and leaf expression (P<0.01); B: Relative expression level of roots and leaves under 150 mmol·L-1 NaCl treatment;* and ** respectively indicated the difference between different time in same organization and 0 h is significant (P<0.05) and extremely significant (P<0.01).
Fig.5 Analysis of MsBBX24 gene relative expression level
图7 WT和转MsBBX24基因拟南芥的幼苗耐盐性分析*和**分别表示转基因拟南芥植株与野生型相比差异显著(P<0.05)和差异极显著(P<0.01),下同。* and ** respectively indicated the transgenic Arabidopsis plants showed significant difference (P<0.05) and extremely significant difference (P<0.01) compared with wild type, the same below.
Fig.7 Salt tolerance analysis of WT and MsBBX24 transgenic Arabidopsis seedlings
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