【作者】 张艳；

【导师】 禤维言；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2014， 硕士

【摘要】 石栗(Aleurites moluccana)是大戟科(Euphorbiaceae)的木本油料植物,其种子油脂含量很高,是一种很重要的生物质能源植物。本试验克隆获得石栗ACCase中BCCP(生物素羧化酶载体蛋白,biotin carboxylase carrier protein)亚基以及BC (biotin carboxylase,生物素羧化酶)亚基的编码基因accB与accC的全长cDNA序列,对其进行生物信息学分析,并通过半定量RT-PCR分析这两个基因在石栗种子不同生长时期中的表达变化,为今后筛选分子标记或研究油脂合成相关酶基因提供参考。1.通过同源克隆法和RACE技术相结合,克隆得到了accB、accC这两个基因的全长cDNA序列,并推导出编码区氨基酸序列。通过NCBI网站及相关软件进行分析比对。结果显示,accB基因全长cDNA序列大小为1170bp,编码区长度为798bp,编码265个氨基酸。accB基因核苷酸序列与基因库中已注册的油桐、麻风树的核苷酸酸序列的同源性依次为：93%和82%,其氨基酸序列与陆地棉、油梨、大豆、苜蓿、麻风树、油桐的BCCP亚基的氨基酸序列同源性分别为：91%、83%、68%、64%、61%、52%；accC基因全长cDNA序列大小长度为1607bp,编码区长度为1119bp,编码372个氨基酸,accC基因核苷酸序列与油桐、麻风树和蓖麻的核苷酸酸序列的同源性分别为：97%、93%和91%,其氨基酸序列与麻风树、陆地棉、烟草、花生、大豆、拟南芥的BC亚基的氨基酸序列同源性分别为：96%、91%、89%、89%、88%、83%。2.采用反转录试剂盒将种子总RNA反转录后,获取了高质量的cDNA。以cDNA为模板,石栗管家基因Actin作为内参,通过半定量RT-PCR分析了石栗accB、accC这两个基因在石栗种子内不同发育时期的表达水平变化。在种子发育过程中accB基因与accC基因表达量大致情况是：六月份>七月份>五月份,同时油脂含量从五月份至七月份三个月间是持续上升的。得出结论：石栗种子油脂含量的升高与石栗accB、accC基因的表达变化有一定相关性。更多还原

【Abstract】 Aleuritesmoluccana is a kind of woody oil plant which belongs to Euphorbiaceae. Its seeds contain high proportion of oil, which makes Aleurites moluccana to be an important biomass energy plant.In this experiment, whole-sequences cDNA of accB gene and accC gene of acetyl coenzyme A carboxylase BCCP subunit and BC subunit were extracted by cloning.And the sequences were analyzed in NCBI bybioinformatics method. Doing the RT-PCR semi-quantitative analysis of the expression of these two genes ofAleurites moluccana seeds in different growth stages is to provide a reference forscreeningmolecular marker or researching related enzymes about fat synthesis。(1) By homology cloning method combined with technology of RACE (cDNA endsRapid Amplification),obtained the full length sequences of accB and accC gene, and deduced their amino acid sequence by Bioinformatics.The BLAST program was used on NCBI website and related sofeware for analyzing and comparing. The result as follow:obtained an1170bp sequence of whole cDNA of accB gene, ORFwas798bp. The homology in nucleotide sequence of GeneaccB with Vernicia fordiiand Jatropha curcas which already registered in the GenBank were as follow:86%and81%. And the homology in amino acid sequence wite BCCP subunit of Vernicia fordii, Jatropha curcas, Medicago truncatula, Glycine max, Persea americana and Gossypium hirsutum andwere:91%,83%,68%,64%,61%, and52%.Obtaining an1607bp sequence of whole sequence of cDNA of accC gene, which contained1119bp ORF.The homology in nucleotide sequence of GeneaccC with Vernicia fordii and Jatropha curcas which already registered in the GenBank were as follow:86%and81%. Andthe homology in amino acid sequence with BC subunit of Jatropha curcas, Gossypium hirsutum, Nicotiana tabacum, Arachis hypogaea, Glycine max and Arabidopsis thalianaandwere:96%,91%,89%,89%,88%, and83%.(2) Taking the seed total RNA as a template, high quality cDNA was obtainedby using reverse transcription kit for seed total RNA transcription. Taking cDNA as a template and Actin about Aleurites moluccana Housekeeping gene as internal reference, expression of geneaccBand gene accC was analysised from Aleurites moluccana at different developmental stages of the seeds by semi-quantitative RT-PCR. In the process of seed development, expression quantity of accB gene rise quickly since may,and then decline slightly in July.But the quantity is higher in July than May. expression quantity of accC gene continue to rise since May to June,and then Rise gently in July.But there were not great difference between June and July. The conclusion:There are certain relationship between seed oil content of Aleurites moluccana and the expression of accB gene and accC gene.The expression quantity of accC gene are higher than accB gene.So result showed that the effect ofaccC gene on Aleurites moluccanafat synthesis may be greater than accB gene.更多还原