枸杞Lb14-3-3c基因的克隆及转化马铃薯的研究
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作者单位:

1.宁夏大学;2.宁夏大学生命科学学院/西部特色生物资源保护与利用教育部重点实验室;3.宁夏气象局科研所

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基金项目:

国家自然科学基金(31360361, 31560418 和 31360025);宁夏优势特色作物现代分子育种重点实验室创新平台专项资金 (2018DPC05019)


Construction of Overexpression Vector of Lb14-3-3c Gene from Wolfberry (Lycium barbarum) and Study of Genetic Transformation to Potato (Solanum tuberosum)
Author:
Affiliation:

1.College of Life Science, Ningxia University/Western Biological Resources Protection and Utilization Lab of National Education Ministry;2.Ningxia Meteorological Bureau Research Institute

Fund Project:

National Natural Science Foundation of China (31360361, 31560418 and 311600025);Innovation Platform Special Funds of Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia (2018DPC05019)

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    摘要:

    在开花植物中,14-3-3蛋白对植物生长发育具有重要的调控作用。本试验利用逆转录PCR( RT-PCR) 技术,从宁夏枸杞宁杞1号花药中克隆了一个14-3-3蛋白家族基因Lb14-3-3c。利用荧光定量PCR技术分析Lb14-3-3c基因在花药发育不同时期的表达特征,构建Lb14-3-3c基因的植物过表达载体 pCambia1305.1-35s-Lb14-3-3c(+)及植物抑制表达载体pCambia1305.1-35s-Lb14-3-3c(-),并用冻融法将构建好的表达载体分别转入农杆菌 GV3101中,继而经农杆菌介导法转化马铃薯紫花白幼茎,并分析了Lb14-3-3c基因在阳性转化苗中的表达情况及叶片淀粉含量。生物信息学分析表明,Lb14-3-3c基因的完整开放阅读框(ORF)长777 bp,具有14-3-3蛋白家族保守结构域,蛋白质分子量为64.14 kD,等电点为4.95,其二级结构主要为α-螺旋。其氨基酸序列与烟草14-3-3c-1的相似度为90.04%,Lb14-3-3c基因编码的蛋白与番茄处于进化树同一分枝上,亲缘关系最近。荧光定量PCR分析表明,该基因在枸杞各器官都有表达,在雄蕊中的表达量最高。在花药发育的各个时期均表达,且在小孢子母细胞时期表达量最高。成功将外源基因Lb14-3-3c载入含强启动子CaMV35s的植物表达载体中,并将该基因的植物过表达载体转化马铃薯,通过表型观察与PCR阳性鉴定得到5株转基因植株,发现转基因植株长势优于野生型植株,苗期野生型与转基因型淀粉含量相差不大,结薯期和成熟期转基因型马铃薯的叶片淀粉含量均高于野生型,且差异显著,且差异显著,本研究为进一步探讨Lb14-3-3c基因对枸杞花药发育过程中淀粉供能的调控提供了参考依据,并且为阐明Lb14-3-3c基因在植物发育过程中的功能及枸杞的分子遗传改良提供了研究基础。

    Abstract:

    In flowering plants, 14-3-3 protein plays an important regulatory role in plant growth and development. In this experiment, a 14-3-3 protein family gene Lb14-3-3c was cloned from the anther of Ningqi No.1 by reverse transcription PCR (RT-PCR). The expression characteristics of Lb14-3-3c gene in different stages of anther development were analyzed by real-time PCR. The plant over-expression vector pCambia1305.1-35s-Lb14-3-3c(+) of Lb14-3-3c gene and plant inhibition were constructed. The vectors were transformed into Agrobacterium GV3101 by freeze-thaw method, and then transformed into Astragalus membranaceus by Agrobacterium-mediated transformation. The expression of Lb14-3-3c gene in positive transformed seedlings and leaf starch content were analyzed. Bioinformatics analysis showed that the complete open reading frame (ORF) of Lb14-3-3c gene is 777bp long and has a conserved domain of 14-3-3 protein family. The molecular weight of the protein is 64.14 kD and the isoelectric point is 4.95. The grade structure is mainly α-helix. The amino acid sequence has a similarity to tobacco 14-3-3c-1 of 90.04%. The protein encoded by Lb14-3-3c gene is on the same branch of tomato as the phylogenetic tree, and has the closest relationship. Real-time PCR analysis showed that the gene was expressed in all organs of the sputum, and the expression was highest in the stamens. It is expressed in all stages of anther development and has the highest expression during microspore mother cell. The exogenous gene Lb14-3-3c was successfully loaded into the plant expression vector containing the strong promoter CaMV35s, and the plant overexpression vector of the gene was transformed into potato. Five transgenic plants were identified by phenotypic observation and PCR positive identification. The growth rate of transgenic plants was better than that of wild-type plants. The content of wild-type and transgenic starch in seedling stage was similar. The starch content of transgenic potato in potato and mature stage was higher than that in wild type. This study provides a reference for the further study of Lb14-3-3c gene regulation of starch supply during the development of anthers, and provides a demonstration of the function of Lb14-3-3c gene during plant development and molecular genetic improvement.

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周丽,张兴,徐 蕊,等.枸杞Lb14-3-3c基因的克隆及转化马铃薯的研究[J].植物遗传资源学报,2019,20(6):1523-1534.

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  • 收稿日期:2019-05-06
  • 最后修改日期:2019-09-22
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  • 在线发布日期: 2019-11-19
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