中国农业科学 ›› 2021, Vol. 54 ›› Issue (22): 4851-4868.doi: 10.3864/j.issn.0578-1752.2021.22.012
收稿日期:
2021-01-25
接受日期:
2021-06-01
出版日期:
2021-11-16
发布日期:
2021-11-19
通讯作者:
段巧红,黄家保
作者简介:
王洁,E-mail: 基金资助:
WANG Jie(),WU XiaoYu,YANG Liu,DUAN QiaoHong(
),HUANG JiaBao(
)
Received:
2021-01-25
Accepted:
2021-06-01
Online:
2021-11-16
Published:
2021-11-19
Contact:
QiaoHong DUAN,JiaBao HUANG
摘要:
【目的】通过对大白菜ACA(Ca2+-ATPase)基因家族鉴定与表达分析,研究其家族基因间的共性与特性,为进一步揭示ACA家族进化关系提供数据支撑,为深入解析BraACAs在低温胁迫、盐胁迫以及自交不亲和方面的功能研究奠定基础。【方法】根据已报道的拟南芥ACA基因家族,同源比对出大白菜ACA基因家族,利用在线软件Expasy预测其分子量、理论等电点等理化性质;采用MEGA 5.0软件构建系统进化树;运用在线软件GSDS 2.0绘制基因结构图谱;TBtools对其染色体定位;McscanX软件进行拟南芥与大白菜ACA家族基因共线性分析;利用在线软件PlantCARE预测大白菜ACA基因家族启动子元件;通过在线工具Pfam和MEME进行蛋白保守结构域分析;利用qRT-PCR技术检测BraACAs在不同组织、非生物胁迫和自交异交授粉后的表达量。【结果】大白菜ACA基因家族有18个基因成员,分布在大白菜10条染色体上;根据进化树关系分成4组,分别包含3、4、4和7个成员;蛋白结构域分析显示,有13个成员包含N端自抑结构域。qRT-PCR结果表明,BraACAs主要在花与果荚中高表达;低温胁迫下,Bra002762与Bra035649表达量总体上调;盐胁迫下,Bra031701表达量显著上调;自交和异交授粉中,Bra003276和Bra024117差异性表达。对Bra002762、Bra035649、Bra031701、Bra003276和Bra024117亚细胞定位分析,发现这些基因均定位在细胞质膜上。【结论】大白菜ACA家族基因蛋白结构均含有4个ACA基因特有的高度保守结构域。该家族在大白菜不同组织中表达模式不同,5个ACA家族基因成员编码蛋白定位于细胞膜上,其中Bra002762、Bra035649、Bra031701与低温和盐胁迫响应有关;Bra003276和Bra024117与自交不亲和性相关。
王洁,吴晓宇,杨柳,段巧红,黄家保. 大白菜ACA基因家族的全基因组鉴定与表达分析[J]. 中国农业科学, 2021, 54(22): 4851-4868.
WANG Jie,WU XiaoYu,YANG Liu,DUAN QiaoHong,HUANG JiaBao. Genome-Wide Identification and Expression Analysis of ACA Gene Family in Brassica rapa[J]. Scientia Agricultura Sinica, 2021, 54(22): 4851-4868.
表1
qRT-PCR引物"
基因名称 Gene name | 上游引物(5´-3´) Forward primer (5´-3´) | 下游引物(5´-3´) Reverse primer (5´-3´) |
---|---|---|
Bra017841 | AAAAGCTTCTGGGTGTTCGTAT | GGTAGCTATCCCAACGATCAAA |
Bra039940 | ACCAGAATTTTGACGTTAAGGC | GAGATAAATTGGAATGCGGCTT |
Bra010605 | GCTGTTGGTTCAGTCGATATTC | GGCTCAACTTTCACCACTTTAC |
Bra010917 | GAAGCTTCAAGATGGTTCTCAC | ATTGTCTCACAAGCAGCTAGAT |
Bra023790 | TCGACAAGCTTACTTCTTCGAT | ATCGCTTTTCGTATTCTTTCCG |
Bra033900 | GTCTGCTACCACTATCTGTACC | TTGATTCTAAACCGGACCAGAA |
Bra023899 | TCAAGCACTAAGGAAAAACGG | ATATTTGCATACACAGAACGGC |
Bra031259 | AATAACGGTGATGGGTAGATCC | ATATTTGCATACACAGAACGGC |
Bra016926 | ATCTCTGACGTCATTGAAGGTT | AATCGCTTTAGCTGTGCTTATG |
Bra003276 | GCTGATGTGATCATAATGGACG | ATACAAAGTTGATGATCAGCGC |
Bra007319 | GTATCCATCGTGAAAAACCGAG | GTTTATATTCTGTCCGTGCACC |
Bra019960 | GTGATTCTGCTGATATTGGCTG | GCTATATTTGCTTGGTAGGCAC |
Bra031701 | TTCTGTCACGCCATTGTCCT | GGTCTATGGCAAGGCAAGGT |
Bra002762 | GAGAGTTCGGACATCATCATCT | GTTAATGATAAGAGCAGCGACG |
Bra035649 | GTAGTATTTACGTCCCAGAGGG | AGAATAGAAGACTGCGATCTGG |
Bra011144 | GCTCATAATACAACTGGCAGTG | GCATCGAAATTCATGCCTAACT |
Bra024117 | CTTGTCGTCCGCTATTTTACTG | TAGGCAAGAGTCAAGGTAACAG |
Bra037404 | CGGAGATAGGCAAGATACAGAG | TGTAGTTGATGATCCAGACGAG |
BraActin2 | CGGTGTCATGGTTGGGAGA | CGTGCTCGATGGGGTACTTC |
表2
部分克隆基因引物序列"
引物名称 Primer name | 正向引物(5´-3´) Forward primer(5´-3´) | 反向引物(5´-3´) Reverse primer(5´-3´) |
---|---|---|
Bra002762-pCAMBIA1300-GFP | ATGACCAGTCCCTTCAAGCCA | GAGTGAACCTTCTCCAGAAGATTTTT |
Bra035649-pCAMBIA1300-GFP | ATGACTAGCCTCTTCAAGCAATCTC | CAAGGCTTCAAAAGTTTGTTGTTT |
Bra031701-pCAMBIA1300-GFP | ATGGAAGACGCTTACGCCAG | CGCCGGTCCTTGGGGAGT |
Bra024117-pCAMBIA1300-GFP | ATGAGTGGTGGACAAGGACAGTT | ACCTGATGAATTCCTTCTCCATCG |
Bra003276-pCAMBIA1300-GFP | ATGTCTAATCTCCTCAAGGATTTTCA | GGCAGAGTCAGATGGACCAGAA |
表3
大白菜ACA基因家族成员信息"
基因ID Gene ID | 理论等电点 pI | 分子量 Molecular weight (Da) | CDS长度 CDS length (bp) | 氨基酸 No. of amino acids (aa) | 亚细胞定位 Subcellular location | 跨膜结构数量 Number of membrane spanning domain | 拟南芥对应ID Arabidopsis corresponds ID |
---|---|---|---|---|---|---|---|
Bra017841 | 5.58 | 110370.20 | 3045 | 1015 | 细胞质膜Plas | 8 | AT4G37640 |
Bra039940 | 5.65 | 110609.40 | 3045 | 1015 | 细胞质膜Plas | 8 | AT2G22950 |
Bra010605 | 5.45 | 112318.20 | 3093 | 1031 | 细胞质膜Plas | 8 | AT4G37640 |
Bra010917 | 5.40 | 110689.40 | 3054 | 1017 | 细胞质膜Plas | 8 | AT1G27770 |
Bra023790 | 8.75 | 112419.20 | 3054 | 1018 | 细胞质膜Plas | 8 | AT3G22910 |
Bra033900 | 8.28 | 112171.60 | 3048 | 1016 | 细胞质膜Plas | 8 | AT3G22910 |
Bra023899 | 6.16 | 119249.50 | 3291 | 1097 | 细胞质膜Plas | 10 | AT3G21180 |
Bra031259 | 6.09 | 119273.50 | 3288 | 1096 | 细胞质膜Plas | 8 | AT3G21180 |
Bra016926 | 5.43 | 112256.90 | 3099 | 1033 | 细胞质膜Plas | 8 | AT2G41560 |
Bra003276 | 6.08 | 111847.50 | 3078 | 1026 | 细胞质膜Plas | 8 | AT3G57330 |
Bra007319 | 5.84 | 111456.90 | 3066 | 1022 | 细胞质膜Plas | 8 | AT3G57330 |
Bra019960 | 5.06 | 113187.20 | 3138 | 1046 | 细胞质膜Plas | 7 | AT1G10130 |
Bra031701 | 5.26 | 118294.80 | 3256 | 1086 | 细胞质膜Plas | 8 | AT1G10130 |
Bra002762 | 8.03 | 116025.10 | 3222 | 1074 | 细胞质膜Plas | 7 | AT5G57110 |
Bra035649 | 7.81 | 116661.70 | 3231 | 1077 | 细胞质膜Plas | 7 | AT5G57110 |
Bra011144 | 6.48 | 116775.40 | 3210 | 1070 | 细胞质膜Plas | 8 | AT5G57110 |
Bra024117 | 6.39 | 115856.20 | 3186 | 1062 | 细胞质膜Plas | 8 | AT4G29900 |
Bra037404 | 5.31 | 115615.30 | 3165 | 1055 | 细胞质膜Plas | 8 | AT4G00900 |
平均数 Average | 6.28 | 114171.00 | 3142 | 1047 | \ | \ | \ |
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