脱落酸(ABA)对植物开花的影响:NCED基因表达、ABA水平与植物开花三者之间的关系
其他题名Abscisic acid affects plant flowering: the relationship among NCED expression, ABA content and flowering
徐平珍
学位类型硕士
导师胡运乾
2007-01-11
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词Nced Aba 开花
摘要植物开花是有花植物生活中一个极其重要而复杂的事件。很多植物生理学研究表明ABA与植物开花有关,会影响植物的开花时间。而且已有研究证明NCED催化的9-顺-环氧类胡萝卜素的裂解反应是ABA生物合成中的关键步骤。因此通过调控NCED基因的表达改变植物体内ABA含量,研究ABA水平与植物开花时间之间的关系是可行的,并且具有一定的理论意义和应用价值。本文对NCED基因表达、ABA水平、植物开花这三者之间的关系进行了研究,主要结果如下: 1. CmNCED基因的克隆 从板栗叶中克隆了CmNCED基因,全长共有1964bp,ORF长1647bp,编码549个氨基酸,5’UTR和3’UTR分别为62bp和229bp。蛋白质分子量为61.5kD,等电点为6.09。用ChloroP 1.1 Server在线预测,CmNCED的氨基酸序列与AtNCED1、PvNCED2和PaNCED2相似,N末端无叶绿体转运肽。对比分析显示CmNCED与豌豆PsNCED1和拟南芥AtNCED1的氨基酸序列有很高的同源性,分别为84%和82%;而与LeNCED1和ZmVP14均只有32%的同源性。CmNCED基因中含有其他已知NCED基因所共有的四个氨基酸保守区以及四个保守的组氨酸。 2. 植物表达载体的构建及烟草的遗传转化 用限制性内切酶Xba I或Xba I和BamH I分别酶切含有CmNCED和LeNCED1基因的克隆质粒以及双元表达载体pCambia2301-101和pCambia1300-221。T4连接酶进行连接后,转化大肠杆菌,即获得了含有目的基因的植物表达载体。通过酶切分析和DNA测序分析确定目的基因的插入方向。共构建了3个正义和3个反义植物表达载体,它们分别为pCambia2301-101-CmF、pCambia1300-221-CmF、pCambia1300-221-LeF、pCambia2301-101-CmR、pCambia1300-221-CmR和pCambia2301-101-LeR。抽提表达载体的质粒,用冻融法转化根癌农杆菌GV3101,叶盘法转化烟草,便得到相应的转基因植株。 3. 转基因植株的检测 抽提转化株的DNA,经特异引物的PCR分析,结果表明NCED基因已经整合到烟草的基因组中。RT-PCR分析表明CmNCED和LeNCED1基因在转录水平获得了表达。用ELISA法对所有转化株的ABA含量进行了测定,结果显示正义转化株的ABA含量没有显著高于对照株,最高的只高于对照25.4%;反义转化株的ABA水平均低于对照株。 4. 转基因植株的抗旱试验和表型观察 对CmNCED基因的转化株的抗旱试验表明在相同的土壤水分条件下,正义转化株相对于反义转化株和对照株具有更强的耐旱能力,反义转化株相对于野生型植株其对干旱更敏感。 转化株的表型观察中发现反义转化株101CmR-20和101LeR-1的生长异常,其株高明显矮于野生型植株,而且叶片数、最大叶的叶长和叶宽均小于野生型和其他转化株。综合其他研究结果,提出了关于ABA与植物正常生长的高低两个阈值的观点。 对CmNCED基因转化株的开花情况的统计结果表明不论是正义的还是反义的转化株露出花苞所需的时间均稍短于对照株,正义转化株的表现更为明显,并对此现象进行了讨论。
其他摘要Floral development is an important and complicated event in the life cycle of flowering plants. Many plant physiological researches suggest that ABA is involved in plant flowering. A key regulated step of ABA biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenases (NCED). Thus it is feasible to study the relationship between ABA level and plant flowering by regulating expression of NCED. In this paper, the relationship among expression of NCED genes, ABA level and plant flowering was discussed. The main results are as follows: 1. Isolation of CmNCED gene A cDNA, CmNCED, was cloned from Chinese chestnut (Castanea mollissima) leaves. The 1964-bp full-length cDNA sequence has a 1647-bp ORF of 549 aa with a 62-bp 5’UTR and 229-bp 3’UTR. The deduced amino acid sequence of CmNCED is a polypeptide of 61.5 kD with an isoelectric point of 6.09. The chloroplast-transit peptide is lacking in N-terminal predicated by ChloroP 1.1 Server online. The sequence of CmNCED is similar to AtNCED1、PvNCED2 and PaNCED2, it shares 84% and 82% identity at the amino acid level with PsNCED1 of pea and AtNCED1 of Arabidopsis, respectively. However, it is only 32% similar to the sequences of LeNCED1 and maize VP14. The CmNCED contains all four conserved sequences and other conserved regions centered on histidine. 2. Construction of plant expression vectors and transformation of wild tobacco Plasmid containing CmNCED ,expression vectors pCambia2301-101 and pCambia1300-221 were digested by Xba I, meanwhile plasmid containing LeNCED1 and the two expression vectors were digested by Xba I and BamH I. After linked by T4 ligase, the vectors were introduced into E. coli. The orientation was identified by restriction enzyme digesting and DNA sequencing. In all, three sense and three antisense plant expression vectors were constructed, and named as pCambia2301-101-CmF、pCambia1300-221-CmF、pCambia1300-221-LeF、pCambia2301-101-CmR、pCambia1300-221-CmR and pCambia2301-101-LeR. The expression vectors were introduced into Agrobacterium tumefaciens strain GV3101 by freezing-thawing method and the transgenic plants were obtained by Agrobacterium-mediated leaf disc transformation. 3. The detection of transgenic plants NCED gene in transgenic tobacco genome was detected by PCR, using specific primers. The expression of CmNCED and LeNCED1 were detected in both sense and antisense transgenic lines by RT-PCR. We measured ABA levels of all transgenic plants under normal growth conditions by ELISA, and found little increase in the sense transgenic plants, the highest one was about 25.4% higher than control. Both CmNCED and LeNCED1 antisense transformants had lower ABA level than control. 4. Drought tolerance and morphological observation of transgenic plants Drought stress experiment indicated that CmNCED sense transgenic tobacco plants had greater drought tolerance comparing with antisense transformants and control, and antisense transformants appeared to be more sensitive to drought stress than wild-type plants under the same soil water content. Two abnormal antisense transgenic plants were found by morphological observation of all transformants. Comparing with control and other transformants, 101CmR-20 and 101LeR-1 had obviously lower height, fewer leaf number, and smaller blade length and width. Combining with other researches, the viewpoint of high and low limens was brought forward. The flowering time of CmNCED transformants was also noted. Either sense or antisense transformants needed fewer days to come out inflorescence than control, especially sense transgenic lines and the possible reasons were discussed.
页数81
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/128
专题昆明植物所硕博研究生毕业学位论文
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徐平珍. 脱落酸(ABA)对植物开花的影响:NCED基因表达、ABA水平与植物开花三者之间的关系[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2007.
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