本文關(guān)鍵詞：煙草NtAAP2基因的克隆及功能研究　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 煙草氨基酸透性酶 基因家族 植物表達載體 煙草遺傳轉(zhuǎn)化 游離氨基酸

【摘要】：氨基酸是植物體內(nèi)至關(guān)重要的有機氮化合物,煙草葉片中氨基酸的含量不僅可以影響植物的生長發(fā)育,還會影響烤煙的品質(zhì)和風(fēng)味。氨基酸轉(zhuǎn)運蛋白參與了植物體內(nèi)氨基酸的轉(zhuǎn)運,對植物正常生長發(fā)育起著重要的作用。植物中氨基酸轉(zhuǎn)運蛋白可以劃分為APCs(the amino acid polyamine choline transporter)和ATFs(the amino acid transporter family)兩大家族,其中氨基酸透性酶(amino acid permease,AAPs)亞家族分屬于ATFs家族,目前植物中關(guān)于AAPs成員的研究較多。本研究對煙草AAPs家族進行了生物信息學(xué)分析,并從普通煙草(紅花大金元)中克隆得到了兩個NtAAP2基因,分別命名為NtAAP2-1和NtAAP2-2,分析了它們的基因結(jié)構(gòu)特征及在煙草組織中的表達模式。構(gòu)建了NtAAP2-1和NtAAP2-2基因超表達載體和RNA干擾載體,進行煙草遺傳轉(zhuǎn)化,并分析了轉(zhuǎn)基因煙草葉片中氨基酸含量的變化,初步研究NtAAP2-1和NtAAP2-2在煙草生長發(fā)育過程中的生物學(xué)功能。主要實驗結(jié)果如下:(1)普通煙草AAPs基因家族分析本研究中,分別從林煙草(Nicotiana sylvestris)、絨毛狀煙草(Nicotiana tomentosiformis)、普通煙草(Nicotiana tabacum)中鑒定出7個、6個、15個AAP基因,并且對煙草中的AAP基因家族進行了多重分析,包括染色體位置、系統(tǒng)發(fā)育關(guān)系、基因結(jié)構(gòu)、預(yù)測蛋白質(zhì)結(jié)構(gòu)以及關(guān)于保守基序分析等等。分析發(fā)現(xiàn)AAP基因家族成員的氨基酸序列均具有很高的一致性。普通煙草中15個AAP基因分布在8條染色體上,并且基因序列均被內(nèi)含子隔開。系統(tǒng)進化分析顯示,AAPs蛋白可以分為4個簇,煙草AAPs蛋白序列主要分布在簇1和簇4中。(2)煙草NtAAP2基因的克隆及分析從普通煙草紅花大金元中成功克隆出了NtAAP2-1和NtAAP2-2基因的編碼區(qū)序列和基因組序列,其中NtAAP2-1編碼序列長為1542 bp,編碼513個氨基酸,蛋白分子量56.99 kDa,等電點9.52;NtAAP2-2編碼序列長為1539 bp,編碼512個氨基酸,蛋白分子量56.79 kDa,等電點9.45。兩個NtAAP2基因的編碼序列相似度達到了94.42%,氨基酸序列的相似度達93.76%,NtAAP2-1和NtAAP2-2均含有一個Aa_trans結(jié)構(gòu)域和12個跨膜結(jié)構(gòu),并且均為疏水蛋白。進化分析和多序列比對結(jié)果表明,NtAAP2-1與NsyAAP2、NtAAP2-2與NtomAAP2分別互為直系同源。表達模式分析結(jié)果顯示,NtAAP2-1和NtAAP2-2基因均在盛花期的莖和根中高表達。(3)煙草NtAAP2基因轉(zhuǎn)基因株系的創(chuàng)制為了研究Nt AAP2-1和NtAAP2-2基因的生物學(xué)功能,分別構(gòu)建了NtAAP2-1、NtAAP2-2基因過表達載體和RNA干擾載體,對煙草進行遺傳轉(zhuǎn)化,得到T1代轉(zhuǎn)基因煙草。(4)T1代陽性轉(zhuǎn)基因煙草葉片游離氨基酸含量測定通過檢測野生型對照煙草和NtAAP2-2轉(zhuǎn)基因煙草葉片中18種游離氨基酸的含量,發(fā)現(xiàn)轉(zhuǎn)基因煙草葉片中游離氨基酸的含量與野生型煙草葉片游離氨基酸的含量相比,Asp、Asn、Glu和Gln的含量發(fā)生改變,說明NtAAP2-2參與了Asp、Asn、Glu和Gln在煙草體內(nèi)的轉(zhuǎn)運。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S572

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