烟草NtAAP2基因的克隆及功能研究

发布时间：2017-12-31 23:10

本文关键词：烟草NtAAP2基因的克隆及功能研究　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：氨基酸是植物体内至关重要的有机氮化合物,烟草叶片中氨基酸的含量不仅可以影响植物的生长发育,还会影响烤烟的品质和风味。氨基酸转运蛋白参与了植物体内氨基酸的转运,对植物正常生长发育起着重要的作用。植物中氨基酸转运蛋白可以划分为APCs(the amino acid polyamine choline transporter)和ATFs(the amino acid transporter family)两大家族,其中氨基酸透性酶(amino acid permease,AAPs)亚家族分属于ATFs家族,目前植物中关于AAPs成员的研究较多。本研究对烟草AAPs家族进行了生物信息学分析,并从普通烟草(红花大金元)中克隆得到了两个NtAAP2基因,分别命名为NtAAP2-1和NtAAP2-2,分析了它们的基因结构特征及在烟草组织中的表达模式。构建了NtAAP2-1和NtAAP2-2基因超表达载体和RNA干扰载体,进行烟草遗传转化,并分析了转基因烟草叶片中氨基酸含量的变化,初步研究NtAAP2-1和NtAAP2-2在烟草生长发育过程中的生物学功能。主要实验结果如下:(1)普通烟草AAPs基因家族分析本研究中,分别从林烟草(Nicotiana sylvestris)、绒毛状烟草(Nicotiana tomentosiformis)、普通烟草(Nicotiana tabacum)中鉴定出7个、6个、15个AAP基因,并且对烟草中的AAP基因家族进行了多重分析,包括染色体位置、系统发育关系、基因结构、预测蛋白质结构以及关于保守基序分析等等。分析发现AAP基因家族成员的氨基酸序列均具有很高的一致性。普通烟草中15个AAP基因分布在8条染色体上,并且基因序列均被内含子隔开。系统进化分析显示,AAPs蛋白可以分为4个簇,烟草AAPs蛋白序列主要分布在簇1和簇4中。(2)烟草NtAAP2基因的克隆及分析从普通烟草红花大金元中成功克隆出了NtAAP2-1和NtAAP2-2基因的编码区序列和基因组序列,其中NtAAP2-1编码序列长为1542 bp,编码513个氨基酸,蛋白分子量56.99 kDa,等电点9.52;NtAAP2-2编码序列长为1539 bp,编码512个氨基酸,蛋白分子量56.79 kDa,等电点9.45。两个NtAAP2基因的编码序列相似度达到了94.42%,氨基酸序列的相似度达93.76%,NtAAP2-1和NtAAP2-2均含有一个Aa_trans结构域和12个跨膜结构,并且均为疏水蛋白。进化分析和多序列比对结果表明,NtAAP2-1与NsyAAP2、NtAAP2-2与NtomAAP2分别互为直系同源。表达模式分析结果显示,NtAAP2-1和NtAAP2-2基因均在盛花期的茎和根中高表达。(3)烟草NtAAP2基因转基因株系的创制为了研究Nt AAP2-1和NtAAP2-2基因的生物学功能,分别构建了NtAAP2-1、NtAAP2-2基因过表达载体和RNA干扰载体,对烟草进行遗传转化,得到T1代转基因烟草。(4)T1代阳性转基因烟草叶片游离氨基酸含量测定通过检测野生型对照烟草和NtAAP2-2转基因烟草叶片中18种游离氨基酸的含量,发现转基因烟草叶片中游离氨基酸的含量与野生型烟草叶片游离氨基酸的含量相比,Asp、Asn、Glu和Gln的含量发生改变,说明NtAAP2-2参与了Asp、Asn、Glu和Gln在烟草体内的转运。
[Abstract]:Amino acids are important organic nitrogen compounds in plants. The content of amino acids in tobacco leaves can not only affect the growth and development of plants. Amino acid transporter is involved in the transport of amino acids in plants. Amino acid transporter in plants can be divided into APCs (APCs). The amino acid polyamine choline transporters and ATFs. The amino acid transporter family. The amino acid permeable enzyme acid per measurement AAPs subfamily belongs to the ATFs family. At present, there are many studies on AAPs members in plants. The bioinformatics analysis of AAPs family in tobacco was carried out in this study. Two NtAAP2 genes, named NtAAP2-1 and NtAAP2-2, were cloned from common tobacco. Their gene structure and expression pattern in tobacco tissues were analyzed. NtAAP2-1 and NtAAP2-2 gene superexpression vectors and RNA interference vectors were constructed for tobacco genetic transformation. The change of amino acid content in transgenic tobacco leaves was analyzed. The biological functions of NtAAP2-1 and NtAAP2-2 during tobacco growth and development were studied. The main results were as follows: 1) the AAPs gene family of common tobacco was analyzed. Nicotiana sylvestris and Nicotiana tomentosiformis, respectively. Seven, six and fifteen AAP genes were identified from Nicotiana tabacum, and the AAP gene family in tobacco was analyzed. These include chromosomal location, phylogenetic relationships, and genetic structure. It was found that the amino acid sequences of members of the AAP gene family were highly consistent. 15 AAP genes were distributed on 8 chromosomes in common tobacco. Go. The gene sequences were separated by introns. Phylogenetic analysis showed that the AAPs protein could be divided into four clusters. The AAPs protein sequence of tobacco is mainly distributed in cluster 1 and cluster 4. Cloning and Analysis of NtAAP2 Gene in Tobacco the coding region and genomic sequence of NtAAP2-1 and NtAAP2-2 genes were successfully cloned from common tobacco safflower Kim Won-Joong. The length of NtAAP2-1 coding sequence is 1542 BP, encoding 513 amino acids, the molecular weight of protein is 56.99 kDa, the isoelectric point is 9.52; The length of NtAAP2-2 coding sequence is 1539 BP, encoding 512 amino acids and the molecular weight of protein is 56.79 kDa. Isoelectric point 9.45. The similarity of coding sequence of two NtAAP2 genes reached 94.42 and the similarity of amino acid sequence reached 93.76%. Both NtAAP2-1 and NtAAP2-2 contained a Aa_trans domain and 12 transmembrane structures, and both were hydrophobic proteins. NtAAP2-1 and NsyAAP2NtAAP2-2 and NtomAAP2 were homologous to each other, respectively. The results of expression pattern analysis showed that NsyAAP2-2 and NsyAAP2-2 were homologous to each other. Both NtAAP2-1 and NtAAP2-2 genes were highly expressed in stems and roots of flowering stage. In order to study the biological function of NT AAP2-1 and NtAAP2-2 gene, the transgenic lines of tobacco NtAAP2 gene were established. NtAAP2-1 ntAAP2-2 gene overexpression vector and RNA interference vector were constructed for genetic transformation of tobacco. T1 generation transgenic tobacco. The content of free amino acids in T1 generation transgenic tobacco leaves was determined by detecting 18 kinds of free amino acids in wild-type control tobacco and NtAAP2-2 transgenic tobacco leaves. It was found that the contents of free amino acids in transgenic tobacco leaves changed compared with those in wild type tobacco leaves. The results showed that NtAAP2-2 was involved in the transport of ASN Glu and Gln in tobacco.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S572

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