【作者】 徐秀红；

【导师】 舒庆尧；

【作者基本信息】 浙江大学 ， 生物物理学， 2009， 博士

【摘要】 植酸是作物种子中含磷最丰富的化合物,占种子干重的1%以上,种子全磷含量的65%-85%和酸溶性肌醇磷酸盐的90%以上,易与K⁺、Ca²⁺、Mg²⁺和Zn²⁺等金属离子螯合形成植酸盐,主要沉积在作物的糊粉层和胚中。植酸及植酸盐不能被人和非反刍动物吸收,影响了微量元素的生物有效性及对蛋白、脂肪和淀粉的吸收;同时,不能被利用的植酸盐随动物粪便排泄进入水系造成水体富营养化。降低种子中植酸的含量,培育低植酸作物,为解决与植酸相关的营养和环保问题开辟了新的途径。在本试验中,利用组织培养技术获得了纯合致死型的低植酸突变体Os-lpa-XS110-3的纯合植株和种子;通过突变基因定位和候选基因序列分析,明确了Os-lpa-XS110-2和Os-lpa-XS110-3两个等位低植酸突变发生在OsMRP5基因,并利用T-DNA插入突变体进行了验证;研究了OsMRP5基因表达的时空特征及突变对转录的影响。主要结论如下:1、Os-lpa-XS110-3的纯合型成熟种子自然发芽时,发芽率低,易发霉腐烂,出苗率在0.1%以下。但是,用四唑法（TTC法）测定突变种子的生活力发现,种子的胚具有活力,并证明可以利用组织培养培育纯合系。对Os-lpa-XS110-3杂合单株自然结实和Os-lpa-XS110-3组培后代不同类型种子单粒重所做的研究表明,与野生型（WT）种子相比,突变型（HIP）种子单粒重显著下降。成熟种子组织培养时,WT种子发芽率显著高于突变型,幼苗的素质也显著要高,表现为幼苗根多,苗高。但是,运用未成熟种子挽救突变体时,野生型和突变型材料的发芽成苗能力没有显著差别。通过组培培养技术获得近300株Os-lpa-XS110-3纯合系。2、通过创建籼粳交定位群体（F₂）,在采用BSA法将低植酸突变基因Os-lpa-XS110-2定位在第3条染色体的基础上,最终将突变基因定位在SSR标记RM14360和RM1332之间大约400Kb之内。In Silico分析表明,该区段存在一个与玉米lpa1基因同源的基因LOC_Os03g04920（OsMRP5）,根据突变体的表型与玉米lpa1的相似性,确定了该基因为Os-lpa-XS110-2突变的候选基因。3、对Os-lpa-XS110-2,Os-lpa-XS110-3两个低植酸突变体的OsMRP5全长序列的测序分析表明,Os-lpa-XS110-2在该基因的第6个外显子上发生了一个C/G-T/A单碱基突变,造成第1156个氨基酸由脯氨酸突变为丝氨酸;Os-lpa-XS110-3则在第1个外显子上发生5个碱基（GGTAG）的缺失,造成从452aa处开始移码突变,并在474aa处形成终止子,极显著地缩短了编码蛋白的长度。同时,对插入到OsMRP5基因的第1个外显子上的T-DNA插入突变体04A-02500的表型及与T-DNA插入的关系进行了分析,发现纯合的插入突变种子与Os-lpa-XS110-3一样,表现为高无机磷含量,不能自然发芽成苗。由此确定了OsMRP5基因在水稻种子植酸代谢过程中的重要作用。4、利用Acitin基因为对照,进行半定量RT-PCR分析,发现OsMRP5基因在水稻幼苗的根、叶鞘和叶片,以及花后7d、14d、21d、28d和35d的籽粒中都有表达,且表达量上基本无差异。同时发现,Os-lpa-XS110-2、Os-lpa-XS110-3在不同组织中OsMRP5的表达水平与亲本秀水110相仿,说明低植酸突变没有改变OsMRP5的RNA的转录水平。相反,T-DNA插入使OsMRP5的转录完全终止。5、对Os-lpa-XS110-2、Os-lpa-XS110-3和T-DNA插入突变体4A-02500的各磷素含量进行了测定,发现与亲本相比,总磷含量基本上不变,没有显著差异。Os-lpa-XS110-2种子的无机磷含量显著增加,约是亲本的4倍,植酸含量下降了33.8%,Os-lpa-XS110-3和4A-02500的纯合突变种子与各自的亲本野生型相比,无机磷含量要高出10倍左右;HPLC分析表明,纯合Os-lpa-XS110-3植酸含量在限值（0.2mg/g）以下未能检测到,纯合的4A-02500突变体只有其亲本的10%,纯合的Os-lpa-XS110-3和4A-02500的无机磷含量和肌醇含量都比他们的野生型显著提高。更多还原

【Abstract】 Phytic acid（PA,myo-inositol 1,2,3,4,5,6-hexakisphosphate） is a ubiquitous component and the most abundant form of P in cereal seeds,accounting for about 65-85%of the total P,more than 90%of acid-soluable inositol phosphate,and 1%of seed dried weight.Phytates,the salt form of PA chelating with cations such as K⁺, Ca²⁺,Mg²⁺,Zn²⁺ and so on,are mostly accumulated in the aleurone and embryo part of cereal seeds.Since PA and phytates are indigestible by humans and non-ruminant animals;they not only affect the bio-availability of minerals and the absorption of protein,fat and starch,but also result in P pollution in water system due to the release of human and animal wastes.Recently,reducing the content of PA in seeds and breeding low phytic acid（lpa） crops have opened a new approach to settle PA-related nutritional and environmental issues.In this study,the plants and seeds of homozygous and lethal lpa mutant Os-lpa-XS110-3 were obtained by tissue culture;we have identified that the OsMRP5 gene is responsible for two allelic lpa mutations in Os-lpa-XS110-2 and Os-lpa-XS110-3,through molecular mapping and sequence analysis of candidate gene. In the mean time,we have validated the gene function using a T-DNA mutant line. Moreover,we have also studied the temporal and spatial expression of the OsMRP5 gene and the effect of the mutations.The major results are as follows:1.When the mature seeds of homozygous Os-lpa-XS110-3 germinate under normal condition,the germination rate is very low;the seeds are easily musty rotten and the rate of emergence is below 0.1%.However,TTC staining indicated that homozygous mutant seeds of Os-lpa-XS110-3 remain viable.Homozygous lines of Os-lpa-XS110-3 were produced by tissue culture using HIP type seeds of Os-lpa-XS110-3.Analysis of seeds derived from heterozygous Os-lpa-XS110-3 plants and the progeny of homozygous Os-lpa-XS110-3 plants rescued by tissue culture,indicated that the weight of single seed of HIP type reduced significantly, compared with the seeds of WT type.The germination rate of WT type seeds is significantly higher than HIP type in mature seeds in vitro culture,and the qualities of seedlings survived from tissue culture are also higher than HIP type,with more roots and taller seedlings.However,the ability of germination of WT and HIP type seeds is consistent in immature seeds in vitro culture.About 300 plants of homozygous Os-lpa-XS 110-3 have been obtained by tissue culture.2.The Os-lpa-XS110-2 mutant gene was first mapped on chromosome 3 through bulked segregation analysis（BSA） of F₂ plants of the japanica/indica population.Subsequently,the mutant gene was mapped to a 400 Kb region between microsatellite markers RM14360 and RM1332 on chromosome 3.In Silico analysis indicated that an orthologous gene LOC_Os03g04920（OsMRP5） of the maize lpal gene was found in this region.According to the phenotype similarity between Os-lpa-XS110-2 and the maize lpal,this gene is chosen as the candidate gene for the Os-lpa-XS110-2 mutation.3.Sequencing results of the OsMRP5 gene of two mutant lines, Os-lpa-XS110-2 and Os-lpa-XS110-3,showed one mutation each occurred in the OsMRP5 gene in the two mutant lines.A single base pair change（C/G to T/A transition） occurred in the sixth exon of Os-lpa-XS110-2,resulting in a change （Pro-Ser） on 1156th aa.A 5-bp（GGTAG） deletion occurred in the first exon of Os-lpa-XS110-3,resulting in frameshift mutation of the gene from 452th aa, generating a premature stop code formation on the 474th aa and significantly shortening the length of encoded protein.Analysis of the phenotype of a T-DNA insertion line,4A-02500,in which a T-DNA was inserted in the first exon of OsMRP5,and relationship with T-DNA insertion indicated that 4A-02500 has the same high inorganic phosphorus phenotype as Os-lpa-XS110-3 and appeared to be homozygous lethal.This evidence shows that OsMRP5 plays an important role in PA metabolism in rice seeds.4.Using an actin gene as a control,semiquantitative RT-PCR analysis showed that OsMRP5 is expressed not only in root,sheath and blade,but also in developing seeds of 7d,14d,21d,28d and 35d after flowering.There were no significant spatial and temporal differences of transcription level.Moreover,the similarity of OsMRP5 gene in different tissues among Os-lpa-XS110-2,Os-lpa-XS110-3 and their parent XS110 indicated that the lpa mutation didn’t change the level of RNA transcription. On the contrary,the transcription of the OsMRP5 gene in the T-NA insertion was disrupted..5.Determination of various P for Os-lpa-XS110-2,Os-lpa-XS110-3 and T-DNA insertion line 4A-02500 showed that compared with their respective wild type lines,there were no significant difference in the total P content.The Pi level of Os-lpa-XS110-2 seeds increased significantly and was about 4 times higher than its wild type,which its PA content reduced by 33.8%.The Pi levels of homozygous seeds of Os-lpa-XS110-3 and 4A-02500 were about 10 times higher than their respective wild type.HPLC analysis indicated that PA-P was not detected（below the limit of quantification,0.2mg/g） in homozygous mutant seeds of Os-lpa-XS110-3.The PA-P content of homozygous seeds of 4A-02500 was only about 10%of the wild type.The Pi content and myo-inositol content in the homozygous seeds of Os-lpa-XS110-3 and 4A-02500 are significantly higher their respective wild type.更多还原