【作者】 斯琴巴拉；

【导师】 陈崇顺；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2000， 硕士

【摘要】 本研究目的在于通过豇豆（Vigna sesquipedalis）特异几丁质酶成熟蛋白基因和菜豆（Phaseolus vulgaris）几丁质酶信号肽基因的克隆、序列分析及其植物表达载体的构建，为以其转化西瓜组织、培育抗枯萎病等真菌病害的西瓜品种奠定重要基础。首先以“早熟二号”豇豆为试材，研究了用不同器官进行提取对DNA提取质量及其PCR扩增效果的影响。结果表明，从营养器官和种子中分别提取的DNA质量及以该DNA分别作模板进行PCR扩增的效果基本相同。接着以PCR扩增豇豆几丁质酶成熟蛋白基因进行PCR条件优化。结果表明，反应物最佳浓度：Mg²⁺为1.5mmol/L,dNTP为0.2mmol/L，引物为0.2μmol/L。然后根据本研究室诱导、纯化、测定的豇豆几丁质酶成熟蛋白N端前10个氨基酸残基的序列和从Genebank中检索到的豇豆属植物（Vigna unguiculata）几丁质酶成熟蛋白C端氨基酸残基的序列，设计、合成了两个引物：P1（5’gaa ttc gag cag tgt gga agc caa gcg ggt ggt gc3’）和P2（5’gga tcc tca gac gat ggg gtg tag att3’），运用PCR技术，从豇豆基因组中分离、扩增了豇豆特异Ⅰ类几丁质酶成熟蛋白基因的全序列；同时分离、扩增了菜豆几丁质酶信号肽基因的序列；并利用T载体分别克隆了这两个基因。测定了该豇豆特异Ⅰ类几丁质酶成熟蛋白基因的全序列，推出了其氨基酸序列，将其与豇豆属植物及菜豆的相应序列作了比较分析。结果表明，豇豆与豇豆属植物Ⅰ类几丁质酶成熟蛋白基因上都有相同的44个酶切位点，而菜豆则具41个酶切位点，且酶切种类及位置等与豇豆均不相同；豇豆与豇豆属植物及菜豆Ⅰ类几丁质酶成熟蛋白的基因序列分别具有98.3％及86.1％的同源性，而其氨基酸序列分别具有97.3％及86.2％的同源性。此外还比较了豇豆、豇豆属植物、菜豆、豌豆（Pisum sativum）、水稻（Oryza sativa）、烟草（Nicotiana tabacum）和小麦（Triticum aestivum）七种植物Ⅰ类几丁质酶成熟蛋白基因及其相应氨基酸序列。结果表明七种基因之间有44.3％以上的同源性，蛋白之间有47.9％以上的同源性，而且在成熟蛋白N端前40个氨基酸序列中分别位于1～13、17～27和36～40位的EQCGSQAGGALCP、CCSQFGWCGST和CQSQC序列区保守性很高，尤其是位于第3、12、17、18、24、31、36、40位的八个半胱氨酸的保守性达到100％。最后将菜豆几丁质酶信号肽基因与豇豆特异几丁质酶成熟蛋白基因重组成嵌合基因，再将其导入pBI121双元载体，构建成完整Ⅰ类几丁质酶基因的植物表达载体。更多还原

【Abstract】 The aim of the study was to lay foundations for transfer of the gene encoding the chitinase ~nd for further acquisition of the Eansgemc plants of Citrullus lanalus resistant to infection by Fusarium oxy.~7porwn f. rnveum, with cloning.and sequence analysis of a chitinasc gene and cobstuction of the plant expression vector. At first, the quality of the DNA extracted from difi~rent oz~mns of the 慫aoshu 2?(V sesquzpedalis) was compared, and the effect of the [INA as template on PCR products was studied, but no difference observed between them, respectively. PCR conditions for amplification of 慫aoshu 2?cbitinase mature protein gene were optimized, and the optimal concentrations arq: Mg~ 1.5ninml/L,dNTP 0.2mmol/L and primer 0.2 ~?mol/L. A pair of primers :P1 (5?gaattcgagcagtgt ggaagc caagcgggtggtgc3? andP2 (5?ggatcGtcgacgatggggtgtagatt3? were designed according to ten N-terminal amino acids ~f chitlnase mature protein from J?gna sesquipedalis, which was induced, purified and sequenced by our lab, and to the C-terminal amino acids of the same protein gene of flgna w2gu1cu?ata from NCBI (ienebank. The particular class I chitinase~ mature protein gene from Vigna sesquz~~edaIis and class I chitinase signal peptide gm~e from Phaseo!us vulgaris were amplified by PCR. The two genes were. cloned into T-vector. The complete sequence of the particular chitinase mature protehi gene from J~?gna sesquz~edalis was determined and its amino acids sequence was deduced. Chitinase mature protein gene sequence and their amino acids sequence from Vigna sesquzpedali.s, ~?gi~a unguiczdata.and Phaseolus vulgarls were compared and analysed. The results showed that 44 Restriction Enzyme sites ~re found in chitinase mature protein gene from Vigna sesquzpedalis and J攊gna ungidculata respectively. Only 41 Restriction Enzyme sites were detetmined in chitinase mature protein gene from Phaseolus vulgaris, and the types and sites of enzymes were different with those from J?gna. The homology between the cbitinase mature protein genes from ~gna sesquipedalis and Vigna unguiculata is 98.3%, tMt from 2 I?gna sesquzpedalis and Phaseolus vulgaris is 86.1%; while the homology between two amino acids sequence of the chitinase mature protein from Vigna sesquipedalis and 1?gna unguiculata is 97.3%, that from Vigna sesquipedalis and Phaseolus vidgaris is 86.2%. Moreover, seven class I chitinase mature protein genes and amino acids sequences from Vigna sesquipedalis, J4gna unguiczdata., Phaseolus vulgaris, Pisum sativum, Oryza sativa,Nicotiana tabacum and Triticum aestivum were compared. The results showed that the gene and amino acid sequence homology between seven species are over 44.3% and 47.90/o, respectively. In addition, conservation of three sequences: EQCGSQAGGALCP, CCSQFOWCGST and CQSQC in the 40 amino acids sequence at the beginning of the N-terminal chitinase mature protein is very high, The conservation of eight Cys at the sites of 3,12,17,18,24,31,36,40 is 100% Finally, the chitinase signal peptide gene from Phaseolus vu?garis and particular chitinase mature protein gene from Vigna sesquipedolis were constructed into one chimera gene. The chimera gene recombinated into pBIl2l binary vector, and the plant expression vector of complete class I chitinase gene was obtained.更多还原