【作者】 王安娜；

【导师】 马凤鸣；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2010， 硕士

【摘要】 大豆(Glycine max (L.) Merr.)是我国重要的粮食和油料作物,含有丰富的蛋白质和脂肪,综合利用价值高,而因为重迎茬问题引起了严重的连作障碍,导致大豆抗逆性降低,产量大幅度下降。近年来,研究表明次生代谢产物的化感效应也是引起连作障碍的重要原因之一,同时次生代谢已成为植物分子生物学研究的热点之一。苯丙烷代谢途径是植物最重要的次生代谢途径之一。肉桂酸-4-羟基化酶(C4H,EC 1.14.13.11),又称反式肉桂酸-4-单氧化酶,催化肉桂酸羟化作用产生4-香豆酸盐,是苯丙烷途径中继L-苯丙氨酸解氨酶(PAL)之后的第二个关键酶。为了从分子水平上揭示大豆C4H的结构特点,并为提高其表达活性提供理论依据,本研究以大豆绥农14为材料,利用RT-PCR技术进行大豆肉桂酸-4-羟基化酶基因mRNA(C4H mRNA);利用PCR技术进行大豆肉桂酸-4-羟基化酶基因组基因(C4H DNA)的分离克隆并结合RACE技术进行C4H基因3’末端基因的克隆。在此基础上,利用生物信息学手段对C4H mRNA推导出的蛋白质序列进行主要结构分析和功能预测。通过上述研究得到以下结果:利用RT-PCR方法成功地从大豆叶片中克隆了肉桂酸-4-羟基化酶基因mRNA(C4H mRNA)(登录号为FJ770468)。获得的C4H mRNA基因长度为1766bp,编码区1521bp,与已公布的C4H mRNA序列(登录号为X92437)的编码区相似性达98.77%,两者只有1个碱基的差异(位于1237位的碱基),并与其它植物的该基因序列具有同源性,应用NCBI上的Blast X工具将C4H cDNA的测序结果推导出的蛋白质序列与已知的C4H蛋白质序列(登录号为Q42797)比较,相似性达99.80%,两者只有1个氨基酸的差异(位于396位的氨基酸),编码一个含有506个氨基酸残基的多肽。运用PCR扩增技术首次克隆了大豆肉桂酸-4-羟基化酶基因组基因C4H DNA(登录号为HM036117)。测序结果表明,C4H DNA含有1个外显子,无内含子,外显子序列与获得的C4H mRNA序列完全一致。结合RACE技术克隆了大豆C4H基因3’末端序列,并将登录号为FJ770468的大豆C4H基因mRNA升级到FJ770468 updata,长度为1799bp。用ExPasy软件包和TOPPRED在线分析C4H的理化特性,结果表明C4H的理论等电点PI=5.30,Mw=39092;C4H是易溶、亲水性较强的蛋白,同时有两个明显的疏水峰,有2个跨膜肽段;并运用DNAMAN6.0软件构建了C4H的系统进化树。通过HNN分析二级结构预测结果显示,C4H的α螺旋(Alpha helix)(Hh):251,49.60%,β折叠(Extended strand)(Ee):52,10.28%,无规卷曲(Random coil)(Cc):203,40.12%;Geno3d模建预测,C4H蛋白中β折叠区有57个折叠,折叠区间有24个α螺旋,此外还有14个卷曲结构。氨基酸序列和结构分析显示C4H蛋白包含了一个保守区,即P450 domain。利用SignalP分析得到信号肽存在几率、信号肽锚定几率均为0.498,切割位点位于28和29位氨基酸之间。更多还原

【Abstract】 The soybean (Glycine max (L.) Merr.) is an significant foodstuff and oil crop in our country, it contains full protein and fat, the valve of multiple utilization is high. The antireversion force and the yield of soybean decrease greatly because of the severe continuous cropping. For the past few years, we learned about the allelopathy of secondary metabolite is one important reason of continuous cropping obstacle from study and research. And the study of secondary metabolism have become one hot spot of molecular bioresearch in plants. Phenylpropanoid metabolic pathway is one of the most important secondary metabolism pathway in plants.trans-cinnamic acid-4-hydroxylase (C4H, EC 1.14.13.11), another name is trans-cinnamic acid-4- mono-oxygenase, it catalyzes trans-cinnamic acid into 4-coumaric acid salt by hydroxylation. It is the second key enzyme in phenylpropanoid metabolic pathway after L- phenylalnine ammonialyase (PAL). In order to reveal the construction features of soybean C4H in the molecular level and provide a theoretical basis of increasing the activity of its expression. Test materials is SN14 in this study, the soybean trans-cinnamic acid-4-hydroxylase mRNA(C4H mRNA)and genome DNA (C4H DNAA) was cloned by RT-PCR and PCR, respectively. In addition, 3’termination sequence of Soybean C4H was cloned by RACE technique. The main structure and function of protein which is translated by C4H mRNA was predicted by bioinformatics analysis. The results were as follows:The trans-cinnamic acid-4- hydroxylase mRNA (C4H mRNA)(Accession Number: FJ770468) was cloned successfully from soybean leaves by RT-PCR. The acquired sequence is 1766bp in length and the sequence in coding region of C4H is 1521bp. Compare with the coding region of C4H mRNA announced(Accession Number: X92437), the resemblance is 98.77%, just one basic group is different(situate 1237position ), the homology of this gene order exists in other plants, the protein translated by the sequencing result compares with the foregone protein (Accession Number: Q42797), the resemblance is 99.80%, just one amino acids is different(situate 396 position ), a polypeptide was encoded by 506 amino acid residues.The soybean trans-cinnamic acid-4- hydroxylase genome DNA (C4H DNA) (Accession Number: HM036117). The sequencing result shows that C4H DNA contain one extron and no intron, the sequence of extron and C4H mRNA acquired are the same, completely.3’termination sequence of Soybean C4H was cloned by RACE technique, and updata information of FJ770468, the new sequence is 1799bp in length.The physical chemistry characteristics of C4H were analyzed on-line by ExPasy and TOPPRED, it shows that theory value of C4H PI and Mw is 5.30 and 39092; respectively; C4H is a soluble, hydrophilic protein, while there are two significant hydrophobic peaks and two transmembrane peptide by TOPPRED, the phyletic evolution tree was constructed by DNAMAN6.0. The secondary structure of C4H various amino acid residues were predicted by HNN, respectively, Alpha helix: 251 is 49.60%, Extended strand: 52 is 10.28%, Random coil: 203 is 40.12%; 3D structure was predicted by Geno3d,it shows that 57 extended strands contained 24 alpha helices inβ-folding area and 14 random coils. According to the amino acid sequence and structural analysis, it shows that C4H protein contains one conserved domain, viz. P450 domain. The existing probability and the anchoring probability of signal peptide were both 0.498 which were analyzed by SignalP, the cleavage site was between 28 and 29 position amino acids.更多还原