植物研究 ›› 2024, Vol. 44 ›› Issue (1): 139-151.doi: 10.7525/j.issn.1673-5102.2024.01.016
王珊珊1, 王瑞1, 樊二勤1, 付鹏跃1, 曲冠证1, 王楠2()
收稿日期:
2023-05-12
出版日期:
2024-01-20
发布日期:
2023-12-27
通讯作者:
王楠
E-mail:nanwang2019@caf.ac.cn
作者简介:
E-mail:nanwang2019@caf.ac.cn。基金资助:
Shanshan WANG1, Rui WANG1, Erqin FAN1, Pengyue FU1, Guanzheng QU1, Nan WANG2()
Received:
2023-05-12
Online:
2024-01-20
Published:
2023-12-27
Contact:
Nan WANG
E-mail:nanwang2019@caf.ac.cn
摘要:
通过鉴定楸树(Catalpa bungei)DELLA家族基因并分析CbuGRAS9的基因功能,为楸树生殖调控性状的遗传改良提供理论依据。基于楸树基因组数据,鉴定并克隆了5个与拟南芥(Arabidopsis thaliana)同源的CbuDELLAs基因;利用ExPASy、SWISS-MODEL、Plant-mPloc、PlantCare等在线工具对CbuDELLAs蛋白进行等电点、蛋白结构、亚细胞定位及启动子顺式作用元件预测;以9-1(Catalpa bungei ‘Luoqiu No.1’)和‘百日花’楸树(Catalpa ‘Bairihua’)为材料,分析了CbuDELLAs基因的表达量差异,并通过异源转化拟南芥证实了CbuGRAS9的分子功能,利用酵母双杂交文库筛选了与CbuGRAS9互作的蛋白。结果表明:5个CbuDELLAs蛋白的氨基酸数目为455~588,蛋白相对分子质量为5.04~6.43 kDa,等电点为4.81~5.14;CbuDELLAs蛋白均含有DELLA和GRAS保守结构域,全部为亲水性蛋白。亚细胞定位预测结果显示CbuDELLAs蛋白均定位于细胞核中。启动子顺式作用元件分析发现,这5个DELLAs基因启动子区均含有参与赤霉素反应的顺式作用元件。qRT-PCR结果显示,‘百日花’楸中CbuDELLAs基因表达量显著高于对照9-1楸,其中CbuGRAS9为差异最显著的基因,CbuGRAS9转基因株系的开花时间被明显推迟。鉴定到与CbuGRAS9互作的蛋白质主要富集在核糖体、氨基酸合成、次级代谢、光合作用、TCA循环等代谢通路。
中图分类号:
王珊珊, 王瑞, 樊二勤, 付鹏跃, 曲冠证, 王楠. 楸树DELLA基因家族生信分析及CbuGRAS9的功能分析[J]. 植物研究, 2024, 44(1): 139-151.
Shanshan WANG, Rui WANG, Erqin FAN, Pengyue FU, Guanzheng QU, Nan WANG. Bioinformatics Analysis of CbuDELLAs Gene Family and Functional Analysis of CbuGRAS9[J]. Bulletin of Botanical Research, 2024, 44(1): 139-151.
表1
CbuDELLAs蛋白的基本信息
拟南芥蛋白名称 Protein names in Arabidopsis thaliana | 楸树蛋白名称 Protein names in Catalpa bungei | 编码区长度 Encoding length/bp | 氨基酸数目 Amino acid number | 等电点 Isoelectric point | 相对分子质量 Molecular weight/kDa | 亚细胞定位 Sub-cellular localization |
---|---|---|---|---|---|---|
AtGAI(AT1G14920) | CbuGRAS22 | 1755 | 585 | 4.81 | 6.41 | 细胞核Nucleus |
AtRGA1(AT2G01570) | CbuGRAS1 | 1767 | 588 | 4.99 | 6.43 | 细胞核Nucleus |
AtRGL2(AT3G03450) | CbuGRAS9 | 1662 | 553 | 4.93 | 6.01 | 细胞核Nucleus |
AtGAI(AT1G14920) | CbuGRAS2 | 1368 | 455 | 5.14 | 5.04 | 细胞核Nucleus |
AtRGA1(AT2G01570) | CbuGRAS32 | 1755 | 584 | 5.08 | 6.40 | 细胞核Nucleus |
表2
CbuDELLAs蛋白的二级结构分析
楸树蛋白名称 Protein names in Arabidopsis thaliana | α螺旋 | β转角 | 无规则卷曲 | 延伸链 Extended strand | ||||
---|---|---|---|---|---|---|---|---|
数量 Number | 比例 Percent/% | 数量 Number | 比例 Percent/% | 数量 Number | 比例 Percent/% | 数量 Number | 比例 Percent/% | |
CbuGRAS1 | 265 | 45.07 | 28 | 4.76 | 240 | 40.82 | 55 | 9.35 |
CbuGRAS2 | 257 | 56.48 | 20 | 4.40 | 130 | 28.57 | 48 | 10.55 |
CbuGRAS9 | 273 | 49.37 | 26 | 4.70 | 197 | 35.62 | 57 | 10.31 |
CbuGRAS22 | 269 | 45.98 | 33 | 5.64 | 228 | 38.97 | 55 | 9.40 |
CbuGRAS32 | 269 | 46.06 | 28 | 4.79 | 235 | 40.24 | 52 | 8.90 |
图4
目的片段克隆及连接到pEASY-T1大肠杆菌菌液PCR检测M.DL5000 DNA Marker;7.阴性对照;A.楸树DELLA基因克隆(1~5.基因CbuGRAS1、CbuGRAS2、CbuGRAS22、CbuGRAS32、CbuGRA9克隆产物);B.pEASY-T1-CbuGRAS1在大肠杆菌菌液中的PCR检测(1~6.pEASY-T1-CbuGRAS1菌液PCR产物);C.pEASY-T1-CbuGRAS2在大肠杆菌菌液中的PCR检测(1~6.pEASY-T1-CbuGRAS2菌液PCR产物);D.pEASY-T1-CbuGRAS9在大肠杆菌菌液中的PCR检测(1~6.pEASY-T1-CbuGRAS9菌液PCR产物);E.pEASY-T1-CbuGRAS22在大肠杆菌菌液中的PCR检测(1~6.pEASY-T1-CbuGRAS22菌液PCR产物);F.pEASY-T1-CbuGRAS32在大肠杆菌菌液中的PCR检测(1~6.pEASY-T1-CbuGRAS32菌液PCR产物)。
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