‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化

发布时间：2018-03-31 04:35

  本文选题：‘云香’水仙　切入点：基因克隆　出处：《西北植物学报》2017年02期

【摘要】：为探究多花水仙ACS基因的序列特征及功能,以‘云香’水仙盛花期花瓣为试验材料,根据‘云香’水仙花朵转录组数据信息,通过RT-PCR方法克隆出1个ACS基因,命名为NtACS1(GenBank KX082936);NtACS1开放阅读框(ORF)长度为552bp,编码183个氨基酸。编码蛋白质分子量约为20.6KDa,理论等电点为6.30,不稳定系数为65.49,属于不稳定的疏水性蛋白。通过qRT-PCR对‘云香’水仙不同时期花瓣和副冠中的NtACS1基因进行了表达分析,得到与‘云香’水仙花朵转录组数据中相同的结果:NtACS1基因在‘云香’水仙花瓣和副冠中的表达都是随着花衰老过程呈现逐渐下降的趋势,且NtACS1基因在花瓣和副冠中的表达峰值都在花苞期,表明NtACS1基因编码的蛋白是在乙烯生物合成途径的系统1发挥催化作用的ACC合成酶。成功构建了NtACS1基因的正义植物表达载体,并通过农杆菌介导法获得8株转基因烟草,PCR和RT-PCR检测显示其中有6株为阳性植株,初步证实NtACS1基因已导入烟草基因组中且在烟草中已表达。该研究结果为进一步分析NtACS1基因的功能和后续转化水仙延长其花期研究奠定了基础。
[Abstract]:In order to study the sequence and function of ACS gene of Narcissus multiflorum, a ACS gene was cloned by RT-PCR from petals of Narcissus in flowering period. Named NtACS1(GenBank KX082936NtACS1, the length of ORF is 552 BP, encoding 183 amino acids. The molecular weight of encoded protein is about 20.6KDa. the theoretical isoelectric point is 6.30, and the instability coefficient is 65.49. it is an unstable hydrophobic protein. The expression of NtACS1 gene in petal and accessory at different stages was analyzed. The same results were obtained from the transcriptional data of 'Yunxiang' Narcissus flowers. The expression of the gene in the petals and accessory crowns of 'Yunxiang' narcissus showed a decreasing trend with the flower senescence. The peak expression of NtACS1 gene in petal and accessory was in flower bud, indicating that the protein encoded by NtACS1 gene was a ACC synthase that played a catalytic role in ethylene biosynthesis pathway system 1. The sense plant expression vector of NtACS1 gene was successfully constructed. By Agrobacterium tumefaciens mediated PCR and RT-PCR analysis, 6 of the 8 transgenic tobacco plants were found to be positive. It was confirmed that the NtACS1 gene had been introduced into the tobacco genome and expressed in tobacco. The results laid a foundation for the further analysis of the function of NtACS1 gene and the subsequent transformation of narcissus to prolong its flowering period.
【作者单位】： 福建农林大学园艺学院;福建农林大学园艺植物遗传育种研究所;厦门大学环境与生态学院;
【基金】：福建省种业创新与产业化工程项目(K8114001B)
【分类号】：Q943.2;S682.21
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本文编号：1689201