【作者】 梁业红；

【导师】 范云六；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2005， 博士

【摘要】 作为供给人体叶酸需求的主要来源之一,植物中的主要作物如水稻、小麦和玉米的叶酸含量很低。对植物叶酸合成途径调控机理的研究,目前仅对GTP环化脱羧酶（GCHI）有较深入的研究。本文拟对植物叶酸生物合成途径中的另外2个酶即二氢叶酸合成酶/多谷氨酰胺叶酸合成酶（DHFS/FPGS）以及二氢喋呤合成酶（DHPS）进行研究,探讨它们在植物（拟南芥）叶酸生物合成中的作用。在植物中DHFS和FPGS分别由不同的基因编码,而在E.coli中则由FolC基因同时编码;DHPS在E.coli中由FolP基因编码,在植物中则由类似的基因编码。将来源于E.coli的FolC和FolP基因分别与酵母CoxⅣ基因的线粒体定位序列连接,并由CaMV 35S启动子驱动。取得了以下主要研究结果: 1.系统地分析了来源于E.coli的FolC基因的作用:（1） Northern blot和Taqman RT-PCR分析表明,来源于E.coli的FolC基因在拟南芥植株中得到了表达。（2） 对FolC转基因纯系C4-3-3、C11-6-8和C12-13-1的幼苗植株和幼嫩叶片的叶酸含量分析表明,这3个纯系幼苗植株的叶酸含量分别为2.23、2.46和2.51 nmol/g鲜重,而对照为1.77 nmol/g鲜重:这3个纯系其幼嫩叶片的叶酸含量分别是5.07、5.54和4.31 nmol/g鲜重,对照为3.09 nmol/g鲜重。因此无论是幼苗植株还是幼嫩叶片,转基因纯系的叶酸含量均显著高于对照。（3） mRNA表达高的纯系C11-6-8,其叶酸含量也高,而mRNA表达较C11-6-8低的纯系C4-3-3和C12-13-1,则其叶酸含量也相应低,因此FolC转基因纯系的叶酸含量与FolC基因的mRNA表达量呈正相关。（4） 对转基因纯系其内源DHFS/FPGS编码基因的mRNA表达的定量分析结果表明,外源FolC基因在幼嫩叶片中的过量表达,并没有促进内源DHFS/FPGS编码基因的表达。 因此外源FolC基因在拟南芥植株中的过量表达,提高了植株的叶酸含量,而且转基因植株叶酸含量的提高是由于外源FolC基因而非内源DHFS/FPGS编码基因作用的结果。 2.分析了拟南芥野生型植株内源DHFS/FPGS编码基因的表达以及植株不同发育时期叶片的叶酸含量。Taqman RT-PCR分析表明:内源不同的DHFS/FPGS编码基因其mRNA表达水平不同,幼嫩叶片中DHFS和FPGS-3的表达量最高;此外,DHFS和FPGS-1、FPGS-2、FPGS-3的总表达量在幼嫩叶片中最高,具有组织特异性。叶酸的测定结果表明,不同发育时期叶片的叶酸含量不同。结合内源DHFS/FPGS编码基因的表达分析以及对叶酸含量的分析,结果显示:野生型植株的叶酸含量与内源DHFS/FPGS编码基因的mRNA表达量呈正相关。 3.FolC转基因植株比野生型植株开花明显提前,但对植株的生长发育没有负面影响,相反FolC单拷贝纯系C11-6-1和C12-13-1的单株结荚数和单株干重还明显高于对照植株。 4.Northern blot分析表明,来源于E.coli的FolP基因在拟南芥植株中得到了表达。对FolP转基系株系的叶酸测定结果显示,独立株系P26、P45和P49的T₃幼苗其叶酸含量分别是2.30、1.63和2.03 nmol/g鲜重,而野生型对照为1.32 nmol/g鲜重,转基因株系的叶酸含量均显著高于对照。 综上所述,本文首次研究了DHFS/FPGS和DHPS的编码基因对提高拟南芥叶酸合成的作更多还原

【Abstract】 Unlike plants and microorganisms, humans can not synthesize folate de novo and depend entirely on their dietary supply. Plant foods are the single most important source of folate. However many plant food sources such as rice, wheat and maize are poor in this vitamin.Though the folate biosynthesis pathway of plants is now largely elucidated, little is known about the control of flux and accumulation of folate. The first two pioneering trails of the regulation of folate biosynthesis foucued on GTP cyclohydrolase I (GCHI). The aim of this study is to elucicate whether the other two enzymes of the folate biosynthesis pathway , i.e. dihydrofolate synthase(DHFS)/folypolyglutamate synthase(FPGS) and dihydropteroate synthase (DHPS) will increased the folate content in A. thaliana through genetic modification. DHFS/FPGS and DHPS in E.coli was encoded by FolC and FolP gene, respectively. Each of FolC and FolP gene was fused to the mitochondria targeting sequence from yeast CoXIV gene, and was promoted by double CaMV 35S, respectively. The main results obtained as follows:1. Systematically study of the FolC gene from E.coli was carried out on transgenic Arabidopsis: (1) The analysis by Northern blot and Taqman RT-PCR showed that the FolC gene from E.coli was expressed in Arabidopsis. (2) Folate measurement was conducted both on seedlings and young green leaves. In seedlings, the folate content of homozygous FolC transgenic lines C4-3-3 、 C11-6-8 and C12-13-1 was 2.23, 2.46 and 2.51 nmol /gfw, respectively, and that of the wild type plants was 1.77 nmol /gfw. While in young green leaves , the folate content was 5.07, 5.54 and 4.31 nmol/gfw, respectively, and that of the wild type plants was 3.09 nmol /gfw. Therefore the folate content of the transgenic line was significantly higher than that of wild type plants both in seedlings and young green leaves. (3) The highest expresser of FolC (C11-6-8) also showed the highest folate content, while line C4-3-3 and C12-13-1, which the expression of FolC was lower than that of C11-6-8, showed a relatively lower folate content. It was thun indicated the mRNA level of FolC gene was correlated with the folate level of FolC transgenic lines. (4) The overexpression of FolC gene did not promote the expression of the endogenous genes encoding DHFS/FPGS in FolC transgenic lines.Therefore the overexpression of foreign FolC gene resulted in the enhancement of folate content in Arabidopsis, and it was proved that the increase folate content was from the contribution of foreign FolC gene but not the endogenous genes encoding DHFS/FPGS .2. The profiling of the expression of the endogenous genes encoding DHFS/FPGS was conducted on更多还原