【作者】 叶红霞；

【导师】 吴殿星； Chengdao Li；

【作者基本信息】 浙江大学 ， 生物物理学， 2010， 博士

【摘要】 植酸是植物体内磷的主要储藏形式,在作物籽粒中65%-85%的磷以植酸形态存在,这些植酸磷难以被猪和家禽等非反刍动物吸收,因而在饲料中常通过添加无机磷以满足家畜对磷的需求,这无疑增加了饲养成本,而且这部分难以消化的植酸磷会以粪尿的方式排出,造成水体的富营养化,引起严重的环境污染。同时,植酸被认为是一种典型的抗营养因子,容易与铁、锌等多价阳离子鳌合成不溶性植酸盐,或与蛋白质、淀粉形成不溶性复合物,限制人或动物对于上述营养元素的吸收利用。因此,培育低植酸作物品种对环境保护和提高作物的营养价值具有重要的意义。在本研究中,通过比较基因组学的方法确定了大麦低植酸突变位点在水稻上的共线性区域,预测了大麦6个低植酸突变的候选基因；利用功能标记将目前在其他作物中克隆的低植酸同源基因在大麦遗传群体上进行了定位；通过电子克隆和实验克隆相结合的方法分析了其中1个大麦低植酸的候选基因。主要结果总结如下：1、根据水稻和大麦同源片段的对应关系,并结合目前已克隆的低植酸基因在水稻上的同源基因的位置,对6个大麦低植酸突变的候选基因进行了预测。lpal-1 (M422)位点在水稻基因组上的对应位置为第4条染色体,在此区域内预测有2个与低植酸相关的基因：OsSultr3;3和OsIPK1。lpa2-1 (M1070)位点在水稻基因组上的对应位置为第6条染色体,预测的与低植酸相关的基因为：OsSultr3;4、OsMRP15、OsMRP12、OsMRP11。M640位点在水稻基因组上的对应位置为第6条染色体,在区域内的低植酸相关的基因为OsMRP11。lpa3-1(M635)和M955位点在水稻基因组上对应于第5条染色体,此区域内无候选基因。M678位点在水稻基因组上对应于第3条染色体,该区间是低植酸相关基因聚集的热点区域。4个基因家族中8个与低植酸相关的基因位于此位点附近,它们是OsMRP5; sulfate transporter (OsSultr2;1 OsSultr2; 2 OsSultr1;1 OsSultr1;2); RINO1; OsITP5/6K-2。2、将目前在其他作物中克隆的低植酸基因的同源基因在大麦的遗传群体上进行了定位。结果发现：HvMIK基因定位在Stirling/Harrington群体的第6条染色体GBM 1066c和Bmac344a之间;HvIPK基因定位在Sahara/Clipper群体第1条染色体上,与awrm1位置相同,此位点与已定位的M955和lpa3-1突变位点非常接近；HvLPA1被定位于大麦第6条染色体上；HvMRP4被定位于大麦第4条染色体上,而M678的突变位点也在大麦4H上；HvST基因定位在Sahara/Clipper群体中第2条染色体标记bcd266和cdo665之间,已定位的lpa1-1突变位点就在这一区域。3、采用电子克隆结合实验克隆的方法,依据水稻OsSultr3;3 (LOC_Os04g55800)基因序列blast获得了大麦HvST基因的EST序列并对其进行拼接,利用PCR克隆了的该基因全长,测定了其核苷酸序列。结果发现突变体lpal-1 (M422)在第12个外显子上发生了一个C→T单碱基突变,造成在644aa处形成终止子,缩短了编码蛋白的长度。此外,利用RACE技术扩增了该基因cDNA3’末端序列,在RNA水平上验证了该单碱基突变。系统进化树分析该基因属于硫酸盐转运蛋白家族第3亚族成员,氨基酸同源性比对显示大麦的HvST基因普遍存在于植物界的不同物种中。更多还原

【Abstract】 Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is the primary storage form of phosphorus (P) in cereal seeds, accounting for about 65-85% the total P. It is is considered to be an anti-nutritional substance in animal feed and human diets, since it has the potential to form very stable complexes with minerals and proteins, which are poorly utilized to monogastric animals due to the absence or insufficient amount of the phytate degrading enzymes in their digestive system. As a result, it contributes to human mineral deficiency, especially in populations throughout the developing world that rely on grains and legumes as staple foods. In addition, it also contributes to the eutrophication of lakes and rivers, since large amounts of phytate-P are excreted to the environment with the animal waste. The development of plant cultivars with low phytic acid content is therefore an important priority.In the current study, we identified putative barley low phytic acid genes via comparative genomics analysis with rice, developed and mapped a set of functional gene markers to previously mapping results, using other crops lpa gene information and their homologous barley ESTs, cloned and alasysed one of the candidate gene by the method of in silico cloning combination with PCR amplification. The major results are as follows:1. The barley lpa1-1 mutation was localized to chromosome 2H. BLASTN analysis showed that nine markers at the 2H terminal region controlling low phytic acid were aligned to the rice genome sequences along the terminal region of rice chromosome 4. The candidate gene for lpa1-1 may be either the sulfate transporter (OsSultr3; 3) or inositol 1.4.5-trisphosphate 3-/6-kinase (OsIPK1) based on the barley and rice comparison. The lpa2-1 is located on barley chromosome 7H. Comparative mapping showed the relevant region of barley was collinear with a region on rice chromosome 6. The multidrug resistance associated protein gene (OsMRP11, OsMRP12 and OsMRP15) and sulfate transporter (OsSultr3; 4) may be the candidate gene for lpa2-1. The lpa3-1 and "M955" are situated on chromosome 1H. Comparative mapping showed the relevant region of barley was collinear with a Phylogenetic relationships among the plant sulfate transporters indicate that the predicted protein of HvST gene from barley and OsSultr3; 3 from rice fall into the group 3 of sulphate transporter gene family.更多还原