生物技术通报 ›› 2024, Vol. 40 ›› Issue (3): 193-201.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0957
收稿日期:
2023-10-10
出版日期:
2024-03-26
发布日期:
2024-04-08
通讯作者:
黄先忠,男,博士,教授,研究方向:植物遗传学与基因组学;E-mail: huangxz@ahstu.edu.cn作者简介:
吴星星,女,硕士研究生,研究方向:作物遗传育种;E-mail: wuxingxing2019@163.com
基金资助:
WU Xing-xing(), HONG Hai-bo, GAN Zhi-cheng, LI Rui-ning, HUANG Xian-zhong()
Received:
2023-10-10
Published:
2024-03-26
Online:
2024-04-08
摘要:
【目的】 PISTILLATA(PI)基因属于典型的Type II型MADS-box基因家族成员,是ABC(D)E模型中的B类基因,在植物发育过程中起着重要作用,但辣椒PI同源基因功能研究未见报道。探索辣椒PI基因功能,为深入研究植物PI同源基因的功能机制奠定基础。【方法】 利用RT-PCR的方法从一年生辣椒(Capsicum annuum L.)花器官的cDNA中克隆PI同源基因CaPI,并通过生物信息学方法分析其理化性质、亚细胞定位、蛋白结构和系统进化关系;利用实时荧光定量PCR(RT-qPCR)技术分析基因在辣椒不同组织中的表达特征;构建植物过表达载体35S:CaPI,通过floral-dipping法转化拟南芥。【结果】 该基因开放阅读框648 bp,编码215个氨基酸,相对分子质量为25.13 kD。氨基酸多重序列比对表明,CaPI蛋白N端含有“MGRGKIEIKRIEN”保守基序。系统进化树分析证实,CaPI与马铃薯、番茄和矮牵牛的PI同源基因亲缘关系较近。RT-qPCR证实CaPI主要在花中表达,在花萼中表达量最高,其次是花瓣,在雄蕊中低表达,而在雌蕊中几乎不表达。在拟南芥中过表达CaPI,与野生型拟南芥(Col-0)相比,35S:CaPI转基因植株表现出莲座叶和分枝数量增多等表型,但不影响花器官的发育。【结论】 辣椒CaPI具有促进植物分枝发育的功能。
吴星星, 洪海波, 甘志承, 李瑞宁, 黄先忠. 辣椒CaPI的克隆与功能分析[J]. 生物技术通报, 2024, 40(3): 193-201.
WU Xing-xing, HONG Hai-bo, GAN Zhi-cheng, LI Rui-ning, HUANG Xian-zhong. Cloning and Preliminary Functional Analysis of CaPI Gene in Capsicum annuum L.[J]. Biotechnology Bulletin, 2024, 40(3): 193-201.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
CaPI-R | 35S:CaPI载体35S:CaPI Vector | |
CaPI-F | ||
qCaPI-R | CTTCTGGGAGGAGGCTATGG | RT-qPCR |
qCaPI-F | AAGATCTCAGACTGCTTGGC | |
qCaUBI3-R | TGTCCATCTGCTCTCTGTTG | RT-qPCR |
qCaUBI3-F | CACCCCAAGCACAATAAGAC | |
qAtactin2-R | CCTGGACCTGCCTCATCATAC | RT-qPCR |
qAtactin2-F | CTGGATCGGTGGTTCCATTC |
表1 本研究使用到的引物
Table 1 Primer information used in this study
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
CaPI-R | 35S:CaPI载体35S:CaPI Vector | |
CaPI-F | ||
qCaPI-R | CTTCTGGGAGGAGGCTATGG | RT-qPCR |
qCaPI-F | AAGATCTCAGACTGCTTGGC | |
qCaUBI3-R | TGTCCATCTGCTCTCTGTTG | RT-qPCR |
qCaUBI3-F | CACCCCAAGCACAATAAGAC | |
qAtactin2-R | CCTGGACCTGCCTCATCATAC | RT-qPCR |
qAtactin2-F | CTGGATCGGTGGTTCCATTC |
图2 CaPI蛋白结构分析 A:CaPI蛋白二级结构;B:CaPI蛋白(左)和拟南芥PI蛋白(右)空间结构对比
Fig. 2 Structural analysis of CaPI protein A: Secondary structure of CaPI protein. B: Comparison of spatial structures between CaPI(left)and AtPI(right)proteins
图4 辣椒CaPI蛋白和其他植物的系统进化树 AT:拟南芥,AT5G20240.1;Ca:辣椒,CaPI、CaMADS74和CaMADS53;SMEL:茄子,SMEL4.1_08g015360.1.01、SMEL4.1_02g021760.1.01;Solyc:番茄,Solyc06g059970.4.1、Solyc02g084630.3.1;Peaxi:矮牵牛,Peaxi162-Scf00922g00026.1、Peaxi162Scf00591g00074.1;PGSC:马铃薯,PGSC0003DMG401007392;TRAES:小麦,TRAES3BF021600020CFD t1、TRAES3BF048900050CFD t1、TRAES3BF068500020CFD t1、TRAES3BF009000010CFD t1;Os:水稻,Os01t0883100-01、Os05t0423400-0
Fig. 4 Phylogenetic tree of the CaPI proteins in C. annuum L. and other plant species AT: Arabidopsis thaliana, AT5G20240.1; Ca: Capsicum annuum, CaPI, CaMADS74 and CaMADS53; SMEL: Solanum melongena, SMEL4.1_08g015360.1.01, SMEL4.1_02g021760.1.01; Solyc: Solanum lycopersicum, Solyc06g059970.4.1, Solyc02g084630.3.1; Peaxi: Petunia hybrida, Peaxi162Scf00922g00026.1, Peaxi162Scf00591g00074.1; PGSC: Solanum tuberosum, PGSC0003DMG401007392; TRAES: Triticum aestivum, TRAES3BF021600020CFD t1, TRAES3BF048900050CFD t1, TRAES3BF068500020CFD t1, TRAES3BF009000010CFD t1; Os: Oryza sativa, Os01t0883100-01, Os05t0423400-0
图5 RT-qPCR分析CaPI的组织表达特征 A:CaPI在辣椒不同组织中的表达特征;B:CaPI在花萼、花瓣、雄蕊、雌蕊中的表达情况。小写字母表示在0.01水平上差异显著。下同
Fig. 5 Tissue expression pattern of the CaPI gene using RT-qPCR A: Expression profiles of the CaPI gene in different tissues of C. annuum L.. B: Expression profiles of the CaPI gene in four tissues: sepals, petals, stamens, and pistils. Lowercase letters indicate significant differences at P=0.01 level. The same below
图6 35S:CaPI转基因拟南芥的表型分析 A:生长30 d的35S:CaPI转基因植株与野生型(Col-0)拟南芥的表型对比,#1和#2代表2个独立的转基因株系;B:开花时莲座叶数目统计;C:开花天数统计;D:开花时分枝数统计;E:RT-qPCR分析CaPI在转基因植株和对照中的相对表达量
Fig. 6 Phenotype survey of the 35S:CaPI transgenic plants in Arabidopsis thaliana A: Comparisons of phenotypes between the 35S:CaPI transgenic lines and wild type(Col-0)plants grown for 30 d. #1 and #2 represent two independent transgenic plants, respectively. B: Statistics on the number of rosette leaves during flowering stages. C: Statistics of flowering times. D: Statistics of branch numbers during flowering stages. E: RT-qPCR analysis of the relative expression level of the CaPI gene
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