【作者】 王玲平；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2004， 博士

【摘要】 十字花科(Cruciferae)植物是中国蔬菜作物中栽培面积最大的一类，在我国农业生产和人民生活中占有非常重要的地位，对十字花科植物种质资源的充分利用一直是人们努力的方向。但长期以来，对于十字花科族、属和种的划分存在很多问题和争议。随着分子生物学和生物信息学的发展，从分子水平阐明十字花科植物系统演化关系成为可能。利用叶绿体基因组、核基因组中的一些编码和非编码基因，以及核基因组中的一些重复序列、基因间隔区和多基因家族等来进行物种演化分类的研究越来越多。细胞色素P450是一个古老的基因超家族，在植物次生物质的代谢合成中起着极为重要的作用。本论文以已知的细胞色素P450基因超家族成员CYP86MF基因的保守区设计引物对十字花科重要蔬菜作物的6个属13个物种进行了同源序列的分离克隆，通过核酸序列的差异比较分析，研究了该基因在不同物种中的进化关系；同时，通过保守引物的PCR扩增和RACE相结合的方法对十字花科植物不同物种的细胞色素P450基因家族成员基因全长进行了分离克隆、鉴定和原核表达的研究，获得如下研究结果： (1)通过PCR从十字花科植物不同物种中扩增到11个可以推导出完整氨基酸序列的同源片段。这些CYP86MF同源片段的核苷酸序列相似性均在80％以上，所推导的氨基酸序列相似性都在70％以上。 (2)以大白菜‘黄芽14’(Brassica campestris pekinensis．cv．Huangya 14)为材料分离克隆到一个细胞色素P450基因，命名为BCCYP86MF5，cDNA全长1854 bp，含1575 bp的完整开放阅读框，编码524个氨基酸，其编码蛋白质的分子量为61.2 kDa、等电点为8.96；碱性氨基酸、酸性氨基酸、疏水氨基酸和极性氨基酸分别占总氨基酸的13.74％、11.64％、36.45％和22.70％；二级结构预测包括N-糖基化位点、依赖于cAMP和cGMP的蛋白激酶磷酸化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、酪氨基酸激酶磷酸化位点、N-豆蔻酰化位点和细胞色素P450的典型区域，半胱氨酸亚铁血红素配体信号区等，α-螺旋和β-折叠分别占47.7％、45.0％；与BCCYP86MF1基因的氨基酸序列同源性达到95.2％，与拟南芥CYP86C4的达到85.9％。该基因DNA与cDNA的序列比较发现没有内含子存在，Northern杂交分析表明其在花蕾中特异表达。 (3)以萝卜‘圆白’(Raphanus sativus cv．Yuanbai)为材料分离克隆到一个细胞色素P450基因，命名为RSCYP86MF，cDNA全长1818 bp，含1581 bp的完整开放阅读框，编码526个氨基酸，推测的蛋白质分子量60.9 kDa、等电点8.686，碱性氨基酸、酸性氨基酸、疏水氨基酸和极性氨基酸分别占总氨基酸的13.12％、11.59％、36.69％和23.19％；推测的蛋白质含有细 摘要胞色素P45O的典烈特征，包含一个细胞色素半肤氨酸亚铁血红素配体信号区(FNAGPRLcIG)，在其氨基酸的N端有一典型的疏水区:另外还含有与BCCYPS石人仔万基因所编蛋自质相同的结构域，仅数目差别。蛋白二级结构中a一螺旋和p折叠分别.!l’45.2%、47.1%:与BcCYp86初下I、Atlgl3150的氨基酸同源性分别为9。%、84%，与拟南芥CYP86家族的所有成员的同源性均人T.38%。该基因无内含子存在，Northem杂交分析表明该基因在花蕾中特异表达。 (4)以诸葛菜(口尽动印内阳g优us vtolace。)为材料分离克隆到一个细胞色素P450基因，命名为口vcyps“成尸，cDNA全长1 876bP，含有1 605bP的完整开放阅读框，编码534个氨基酸，所编码的蛋白质分子量为61.4切a、等电点为6.90，其碱性氨基酸、酸性氨基酸、疏水氨基酸和极性氨基酸分别占总氨基酸的12.35%、12.73%、37.08%和23.59%:与BCCYP86人仔万和尺占CYPS石初下相比，所编码的蛋白质二级结构中无cAMP和cGMP的蛋白激酶磷酸化位点，其它结构域的数目也不相同，a一螺旋和p折叠分别占44.9%，46.3%;与CYPS‘cJ、CYPS石C4和BCCYP86材下I的核昔酸序列相似性分别为86%、84%和83%，而氨基酸相似性依次为84%、79%和73%，与拟南芥CYP86家族成员氨基酸序列的相似性最低也达38%。该基因无内含子存在，Northem杂交分析表明该基因在花蕾中特异表达。 (5)将BCCYP86材下I和天客CYPS石初下基因转化到大肠杆菌BLZI中诱导表达，发现T-28℃、1 mmol·L’，的IPTG、诱导20h的条件下，PGEx一BCcyp86叼Fl、PGEX.尺刃了尸召‘初下两个重组质粒均有约80 kDa的蛋白质特征条带出现。 (6)C钾86MF同源基因片段在13个物种中的序列分析表明:它们的核昔酸差异为1 .0%一23.6%，序列差异较适中，适于系统发育分析的研究。除及少CYPS石初下外，属内核酸序列的差异为1.0%一5.7%，氨基酸序列的为2.6%~7.3%，而属间两者的差异分别为5.6%一22，5%和7.3%一31.2%，物种属间核酸和氨基酸序列的差异比属内要大的多.核营酸的替换土要以转换为土，表现出高的转换偏倚。用NJ法和MP法构建的系统进化树具有完全相同的拓扑结构，系统进化树可分为3个大的分支，诸葛菜和芥菜归为一个亚群后再与莽菜聚为一类，白举置信水平值为BCL七引%;拟南芥和高掉菜聚为一个支系，BCL全88%;其余的物种归为一类。演化更多还原

【Abstract】 In view of the fact that Cruciferae crops not only account for the largest growing area of vegetable crops but also play an important role in agricultural activities and lives of people in China, it has been our objective that their germplasm resources were fully utilized. However, there have been many of ambiguities and divergences for the taxa of family, genus and species in Cruciferae for a long time. With the developments of molecular biology and bioinformatics, it is possible to elucidate the phylogenetic evolutionary relationship of Cruciferae at the molecular level. It was well known that studies on evolutions and taxa of species have been more and more fashionable by means of either some encoding and non-encoding genes of cpDNA and nrDNA or repeat sequences, ITS, and multiple genes families of nrDNA. Cytochrome P450 play a critical role in plant secondary metabolites synthesis as an ancient gene superfamily. In this paper, phylogenetic relationship of 13 species involved in 6 genera of Cruciferae were carried out through both the clones of homologous sequences with the primers designed on the basis of conserved regions of CYP86MF gene in cytochrome P450 gene superfamily and the differential analyses of them. Meanwhile, complete sequences of some genes in cytochrome P450 gene superfamily were isolated and identified by SMART PCR-RACE strategy, and expressed in E. colt. The results were as follows:(1) Isolated by PCR from 11 species of Cruciferae, eleven homologous gene segments that deduced amino acids were identities of over 80% at nucleotide sequence level and similarities of over 70% at amino acid sequence level.(2) The cytochrome P450 gene named BCCYP86MFS and isolated from Brassica campestris ssp. pekinensis cv. Huangyal4 was complete nucleotide sequence of 1 854 bp with a nucleotide ORF (1575 bp), which encoded a protein consisting of 524 aa with molecular weight of 62.2 kDa and pI of 8.96. Strongly basic (+) amino acids, strongly acidic (-) amino acids, hydrophobic amino acids and polar amino acids of the protein were 13.74%, 11.64%, 36.45% and 22.70% respectively, and predicted secondary structure of the protein revealed many conserved domains such as N-glycosylation site, protein kinase C phosphorylation site, casein kinase II phosphorylation site, N-myristoylation site, cAMP- and cGMP-dependent protein kinase phosphorylation site, tyrosine kinase phosphorylation site and a cytochrome P450 cysteine heme-iron ligand signature which was typical of cytochrome P450. a -helix and B -sheet of the protein is 47.7%, 45.0% respectively. Compared with cDNA, the BCCYP86MF5 genomic DNA being similarities of 95.2%and 85.9% to BCCYP86MFI and CYP86C4 at amino acid sequence level had no intron. The result of the Northern hybridization showed that it was expressed only in floral buds.(3) A full-length cDNA sequence of RSCYP86MF gene as one of cytochrome P450 gene superfamily from Raphanus sativus cv. Yuanbai was composed of 1 818 bp with a nucleotide ORF (1 581 bp), which encoded a protein of 526 aa with molecular weight of 62.2 kDa and pI of 8.96. Strongly basic (+) amino acids, strongly acidic (-) amino acids, hydrophobic amino acids and polar amino acids of the protein were 13.12%, 11.59%, 36.69% and 23.19% respectively. The secondary structure of this protein speculated had a typically conserved domain of cytochrome P450 cysteine heme-iron ligand signature (FNAGPRLCIG) and a hydrophobic domain at N-terminal of amino acid. In addition to the numerary variance, RSCYP86MF had the same domains as BCCYP86MF5 for the protein structure. Amino acid sequence of the protein with 47.7% of a -helix and 45.0% of B -sheet showed identities of 90%, 84% and over 38% to BCCYP86MF1, Atlgl3150 and every member of CYP86 family, respectively. The RSCYP86MF gene had no intron. The result of the Northern hybridization showed that it was expressed only in floral buds.(4) A complete cDNA sequence of OVCYP86MF gene as one of cytochrome P450 gene superfamily from Orychophrogmiis violaceus showed 1更多还原