生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 140-147.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0163
收稿日期:
2022-02-11
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
杨宏亮,男,硕士研究生,研究方向:植物分子设计育种;E-mail:基金资助:
YANG Hong-liang(), YUAN Zhen, QIAN Xu-jia-zhi, XU Da-wei()
Received:
2022-02-11
Published:
2022-10-26
Online:
2022-11-11
摘要:
类硫素基因Thil(Thionin-like)在植物防御系统中具有抗病原微生物的活性,然而对它的功能却不清楚。通过对Thil基因家族在大麦中的序列组成及基因结构、染色体分布、蛋白保守结构域、系统进化树、基因启动子元件、亚细胞定位和表达谱分析,为其生物学功能研究提供借鉴。共鉴定出7个大麦Thil基因,这些基因分布于1、2、4、5和6号染色体上;基因结构分析均表明,除Thil4存在一定的差异外,其它家族成员序列都较为保守;通过系统进化树可以看出大麦Thil基因分为4个亚家族;表达谱分析结果显示,在大麦不同组织中,Thil基因的表达量有所不同,总体来看,Thil基因在根和种子中的表达量极低,在幼叶中表达量最高。Thil家族不同成员之间的蛋白亚细胞定位结果也不一样,Thil1和Thil2主要定位于细胞膜、细胞核和细胞质上,Thil3和Thil7定位于细胞膜上,Thil6定位于叶绿体。Thil基因与植物的保护和防御机制有关,在幼叶中能够激发植物保护机制的元件响应最多,荧光定量PCR实验结果显示,Thil基因在幼叶中的表达量最高,在亚细胞定位中可以发现不同的成员之间在进化过程中分别参与了不同的发育学过程。
杨宏亮, 袁桢, 钱徐佳志, 徐大伟. 大麦Thionin-like基因家族基因表达谱分析[J]. 生物技术通报, 2022, 38(10): 140-147.
YANG Hong-liang, YUAN Zhen, QIAN Xu-jia-zhi, XU Da-wei. Expression Profile Analysis of Thionin-like Gene Family in Barley[J]. Biotechnology Bulletin, 2022, 38(10): 140-147.
基因名称Gene | 上游引物Forward primer(5'-3') | 下游引物Reverse primer(5'-3') |
---|---|---|
Thil1 | TCAGATTTTCACAAACCCGA | GACCGATTTGACTCAGACCA |
Thil2 | TCTCAGCCGTTGCCATCCTC | CCGCTCGCTCTCTACCTCAC |
Thil3 | TAAAGCCAAGAAGCCAACCA | CATTACAGAAACGGGCACAT |
Thil4 | CAAGCAACTCTCAAGAACAC | TTACAGTTACAGGCACACGA |
Thil5 | ATCTTAGAGCCATTCATTTC | TGACATATACTGGTCTTGGA |
Thil6 | ATAAATAGGAGCCTCGTGTT | CATTACAGAAACGGGCACAT |
Thil7 | CACCCAACCCTGACACACTC | CAGCAACTCTGAGGACGAAC |
表1 qRT-PCR引物序列
Table 1 Sequences of qRT-PCR primers
基因名称Gene | 上游引物Forward primer(5'-3') | 下游引物Reverse primer(5'-3') |
---|---|---|
Thil1 | TCAGATTTTCACAAACCCGA | GACCGATTTGACTCAGACCA |
Thil2 | TCTCAGCCGTTGCCATCCTC | CCGCTCGCTCTCTACCTCAC |
Thil3 | TAAAGCCAAGAAGCCAACCA | CATTACAGAAACGGGCACAT |
Thil4 | CAAGCAACTCTCAAGAACAC | TTACAGTTACAGGCACACGA |
Thil5 | ATCTTAGAGCCATTCATTTC | TGACATATACTGGTCTTGGA |
Thil6 | ATAAATAGGAGCCTCGTGTT | CATTACAGAAACGGGCACAT |
Thil7 | CACCCAACCCTGACACACTC | CAGCAACTCTGAGGACGAAC |
Gene | Forward primer(5'-3') | Reverse primer(5'-3') |
---|---|---|
Thil1 | ATGGCGTCGTCGCAGAAGTTCTTC | ACATGGCTTGGTGCACATGCACT |
Thil2 | ATGGAGTCATCACACAAGCTTTTC | GCACTGCTTGGTGCACATGCAGCT |
Thil3 | ATGGCACCCAGCAAGAGTATTAAG | GGCTTCAACAGACTGAATGACTG |
Thil6 | ATGGCACCCAGCAAGAGTATTAAG | GGCTTCAACAGACTGAATGACTGC |
Thil7 | ATGGAGGCTTCACGCAAGCTCCTC | GTAGCGGCAGTGGGTGGTGCAGA |
表2 Thil基因克隆引物序列
Table 2 Primer sequences for Thil gene cloning
Gene | Forward primer(5'-3') | Reverse primer(5'-3') |
---|---|---|
Thil1 | ATGGCGTCGTCGCAGAAGTTCTTC | ACATGGCTTGGTGCACATGCACT |
Thil2 | ATGGAGTCATCACACAAGCTTTTC | GCACTGCTTGGTGCACATGCAGCT |
Thil3 | ATGGCACCCAGCAAGAGTATTAAG | GGCTTCAACAGACTGAATGACTG |
Thil6 | ATGGCACCCAGCAAGAGTATTAAG | GGCTTCAACAGACTGAATGACTGC |
Thil7 | ATGGAGGCTTCACGCAAGCTCCTC | GTAGCGGCAGTGGGTGGTGCAGA |
基因 Gene | 基因ID Gene ID | 染色体 Chromosome | 氨基酸数目 Number of amino acids | 分子量 Molecular weight/kD | 理论等电点Theoretical pI | 亲水性总平均值 Grand average of hydrophilicity | 脂肪族氨基酸指数 Aliphatic index |
---|---|---|---|---|---|---|---|
Thil1 | HORVU1G007680 | 2 | 231 | 18.83 | 5.3 | 0.748 | 19.05 |
Thil2 | HORVU1G007650 | 2 | 227 | 18.53 | 5.3 | 0.719 | 17.18 |
Thil3 | HORVU1G112060 | 5 | 279 | 23.23 | 5.31 | 0.658 | 24.73 |
Thil4 | HORVU1G097030 | 5 | 300 | 24.96 | 5.20 | 1.055 | 19.33 |
Thil5 | HORVU1G002760 | 1 | 414 | 34.23 | 5.22 | 0.798 | 25.60 |
Thil6 | HORVU1G001100 | 6 | 348 | 29.08 | 5.27 | 0.726 | 26.72 |
Thil7 | HORVU1G082390 | 4 | 249 | 20.62 | 5.23 | 0.911 | 12.45 |
表3 大麦类硫素基因家族成员的基因信息
Table 3 Basic information of Thil family genes from barley
基因 Gene | 基因ID Gene ID | 染色体 Chromosome | 氨基酸数目 Number of amino acids | 分子量 Molecular weight/kD | 理论等电点Theoretical pI | 亲水性总平均值 Grand average of hydrophilicity | 脂肪族氨基酸指数 Aliphatic index |
---|---|---|---|---|---|---|---|
Thil1 | HORVU1G007680 | 2 | 231 | 18.83 | 5.3 | 0.748 | 19.05 |
Thil2 | HORVU1G007650 | 2 | 227 | 18.53 | 5.3 | 0.719 | 17.18 |
Thil3 | HORVU1G112060 | 5 | 279 | 23.23 | 5.31 | 0.658 | 24.73 |
Thil4 | HORVU1G097030 | 5 | 300 | 24.96 | 5.20 | 1.055 | 19.33 |
Thil5 | HORVU1G002760 | 1 | 414 | 34.23 | 5.22 | 0.798 | 25.60 |
Thil6 | HORVU1G001100 | 6 | 348 | 29.08 | 5.27 | 0.726 | 26.72 |
Thil7 | HORVU1G082390 | 4 | 249 | 20.62 | 5.23 | 0.911 | 12.45 |
图6 不同组织中Thil基因的表达水平 RT:根;LF:叶;SM:茎;SG:两周大小幼苗;SD:种子
Fig. 6 Expression levels of Thil gene in different barley tissues RT:Root. LF:Leaf. SM:Stem. SG:Two weeks old seedling. SD:Seed
图7 Thil在亚细烟草中的亚细胞定位分析 所选的Thil-GFP融合蛋白以及GFP空载作为对照在烟叶中独立瞬时表达并在共聚焦显微镜下成像,Bar=20 μm
Fig. 7 Subcellular localization of Thil in barley The selected Thil-GFP fusion protein as well as GFP-empty as the control were independently transiently expressed in tobacco leaves and imaged under a confocal microscope. Bars=20 μm
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