【作者】 李长生；

【导师】 贺浩华；

【作者基本信息】 江西农业大学 ， 作物遗传育种， 2020， 博士

【摘要】 叶色突变体不仅是研究叶绿素合成、调控和降解以及叶绿体的发育和叶绿体-细胞核的信号传递的理想材料,也是水稻遗传育种的特异基因资源。本研究在水稻品种“华丝占”的种植过程中,在大田条件下发现了数株白条纹叶色突变体,该突变体的第3个分蘖的叶片呈白条纹状(叶脉和茎秆呈正常绿色),而该植株的主茎和第1、第2分蘖的茎秆、叶脉和叶片呈正常绿色。以该水稻突变体为材料,电镜观察和比较了主茎、第1个分蘖、第2个分蘖的正常绿叶与第3个分蘖的白条纹叶的形态与细胞学特征差异;测定并分析了其正常绿叶与白条纹叶的的叶绿素含量与光合作用速率差异;利用RNA-seq技术分析了正常绿叶与白条纹叶的转录组差异;利用iTRAQ技术分析了正常绿叶与白条纹叶的蛋白质组差异;取得结果如下。(1)突变体的正常绿叶与白条纹叶相比,正常绿叶的叶绿体细胞数量明显多于白条纹叶的叶绿体细胞的数量,正常绿叶的叶绿体细胞呈现更浓的绿色且形态规则,而白条纹叶叶绿体细胞的绿色更淡且形态不规则。(2)突变体正常绿叶的叶绿素a和叶绿素b含量分别为2.81 mg/g和2.02 mg/g,而白条纹叶的叶绿素a和叶绿素b含量则分别为1.61 mg/g和1.32 mg/g。正常绿叶的光合作用速率平均为42μmol/m~2·S,而白条纹叶的光合作用速率平均为28μmol/m~2·S。正常绿叶的叶绿素含量与光合作用速率显著高于白条纹叶的叶绿素含量与光合作用速率。(3)转录组分析结果显示,突变体正常绿叶与白条纹叶之间存在1,070个差异表达基因;相对于正常绿叶而言,在白条纹叶中存在691个下调表达基因、379个上调表达基因。这些差异表达基因中,有12个差异表达基因极显著富集在核糖体途径(Ko03010)的30S小亚基核糖体蛋白S8与S17,以及核糖体50S大亚基核糖体蛋白L6、L12、L18、L24、L29和L34,有3个差异表达基因极显著富集在光合作用途径(Ko00195)的铁氧化还原蛋白和ATP酶亚基I,有12个差异表达基因极显著富集在苯丙素生物合成途径(Ko0094)的过氧化物酶3、过氧化物酶4、过氧化物酶72、β-葡萄糖苷酶7、β-葡萄糖苷酶25、β-葡萄糖苷酶33、4-香豆酸-CoA连接酶1基因、细胞色素P450、苯丙氨酸氨裂解酶、肉桂醇脱氢酶5和肉桂醇脱氢酶8D。同时,编码β型蛋白质的基因在白条纹叶中呈现上调表达,而编码α型蛋白质和GDP、UDP异构酶的基因在白条纹叶中均表现下调表达。(4)蛋白质组分析结果显示,突变体正常绿叶与白条纹叶之间存在156个差异表达蛋白质。这些差异表达的蛋白质中,有14个蛋白质与核糖体Ribosome蛋白有关,其中10个蛋白质在白条纹叶中呈现下调表达、4个蛋白质在白条纹叶中呈现上调表达。KEGG富集分析显示,有8个差异表达蛋白的编码基因富集在碳代谢途径、另有8个差异表达蛋白的编码基因富集在光合作用途径、还有8个差异表达蛋白的编码基因富集在核糖体途径、有5个差异表达蛋白的编码基因富集在光合器官中的碳固定途径,各有4个差异表达蛋白的编码基因富集在氨基酸生物合成途径与乙醛酸与二羧酸代谢途径以及氧化磷酸化途径。研究结果表明:该突变体的核糖体在基因转录和蛋白质翻译过程中的变异或修饰,导致核糖体的非正常生物合成而功能异常,从而造成光合作用速率下降,最终影响与叶片颜色有关的苯丙素类物质、糖类和淀粉、谷胱甘肽等物质的代谢,而使水稻叶片失绿。水稻粒型和粒重以及灌浆速率是产量形成的重要指标,挖掘并克隆与水稻粒型和粒重以及灌浆速率相关的QTLs,对于确保水稻稳产、高产具有重要意义。本研究以籼稻五丰B和籼稻昌恢T025构建的重组自交系群体为材料,在2018年海南和2019年南昌这两种环境下对粒型(粒长、粒宽、长宽比和粒厚)和粒重进行了QTL定位;同时在2019年和2020年中稻这两种环境下进行了灌浆速率的QTL定位。取得如下结果:(1)在2018年海南和2019年南昌这两种环境条件下,共检测到了32个与水稻粒型和粒重相关的QTLs;两种环境重复检测到了6个QTLs:千粒重qTGW2、粒长qGL12、粒宽qGW2.2和qGW5、长宽比qLWR5,粒厚qGT4.2,这些QTLs分别分布在水稻第2、12、5和4号染色体上。(2)在2019年南昌中稻和2020年南昌中稻这两种环境下,共检测到了6个与灌浆速率相关的QTLs,其中花后1-5 d这个阶段检测到了qGFR1.1,花后5-10 d这个阶段检测到了3个QTLs:qGFR3、qGFR5和qGFR7.2,花后10-15 d这个阶段检测到了2个QTLs:qGFR1.2和qGFR7.1;两年的环境下,未能检测共同的QTLs,推测环境条件对水稻灌浆影响较大。更多还原

【Abstract】 Leaf discolored mutant is ideal research material for chlorophyll biosynthesis,regulation,degradation,and for chloroplast development and signal conduction of chloroplast-nucleus as well as special genetic resource for rice breeding.Several white stripe leaves(wsl)of rice mutant were obtained in present study,which leaves of the main stems,the first seedlings and the second seedlings are normal green color while leaves of the third seedlings are wsl except the leaf vein and stalk,from the rice cultivar Huasizhan at paddy conditons.In present study,the morphologic and cellular traits were observed,and chlorophyll contents and photosynthesis rate were determined and compared between the wsl and normal green leaf(lgl)from the rice mutant.The transcriptomes were analyzed and compared between the wsl and normal green leaf(lgl)from the rice mutant,using the RNA-seq method.The proteomes were analyzed and compared between the wsl and normal green leaf(lgl)from the rice mutant,using the iTRAQ method.The results are as follow.(1)The wsls that were generated from the third seedling exhibited color deficiency and were almost white in color,except the nervure region,whereas the leaves of the major stems and other seedlings maintained lgls.The chloroplasts in the wsl were thin,and chloroplast biogenesis was apparently blocked,and abnormal structures were observed in the wsls compared to the lgls.(2)The chlorophyll a and b contents were 2.81μg/g and 2.02μg/g respectively in the lgls while the chlorophyll a and b contents were 1.61 mg/g and 1.32 mg/g respectively in the wsls.The photosynthesis rates were 42μmol/m~2·S and 28μmol/m~2·S in the lgls and wsls,respectively.The chlorophyll contents and photosynthesis rates were showed significant lower in the wsls than that in the lgls of the rice mutant.(3)From the transcriptomic analyzed results,a total of 1,070 differential expressed genes(DEGs)were detected between the wsls and lgls,with 691 downregulation and 379upregulation in the wsls comparing to the lgls.Among the DEGs,twelve DEGs significantly enriched in ribosome 30S small subunit S8 and S17,and ribosome 50S large subunit L6、L12、L18、L24、L29 and L34 in the ribosome pathway(Ko03010),and the 12DEGs showed downregulated in the wsls,compared to the lgls;Three DEGs significantly enriched the ferredoxins and the ATPase subunit I of photosynthesis pathway,and the 3DEGs were downregulated in the wsls compared to the lgls;The other 12 DEGs significantly enriched in the phenylpropanoid biosynthesis pathways of peroxidase 3,peroxidase 4,peroxidase72,and beta-glucosidase 7,beta-glucosidase 25,beta-glucosidase33,4-coumarate--CoA ligase 1,cytochrome P450 and phenylalanine ammonialyase were upregulated,whereas cinnamyl alcohol dehydrogenase 5,dehydrogenase 8D was downregulated in wsl,compared to the lgl.(4)After proteomics analysis,a total of 156 differential expressed proteins(DEPs)were determined between the wsls and lgls.Among the DEPs,14 DEPs invovled in Ribosome biosynthesis,with 10 DEPs downregulation and 4 DEPs downregulation;eight DEPs involved in carbon metabolism(ko01200);another 8 DEPs involved in Photosynthesis(ko00195);the other 8 DEPs involved in Ribosome(ko03010);Five DEPs involved in carbon fixation in photosynthetic organisms(ko00710);the KEGG pathways of Biosynthesis of amino acids,glyoxylate and dicarboxylate metabolism,and oxidative phosphorylation were enriched 4 DEPs each.The results suggested that the variation or modifications of the genes encoding ribosome during gene transcript and translation process leads to abnormal biosynthesis and abnormal biological function of ribosome,resulting in lower chlorophyll contents and photosynthesis rate,and final abnormal metabolism on phenylpropanoid,sugar,starch and glutathione,resulting in leaf discolor in rice mutant.Rice grain shape,grain weight and grain-filling rate are important indexes for rice yield establishment.The QTLs for these indexes are play important role for stable yield and high yield of rice.In present study,the RILs from Wufeng B/Changhui T025 were used as plant materials under Hainan in 2018 and Nanchang in 2019,and the QTL related to grain shape including grain length,grain width,grain length/width ratio and grain thickness,were located in rice genome;the QTL related to grain-filling rate were also located in mid-season at Nanchang in 2019.The results are as follow:(1)Under the environments of Hainan in 2018 and Nanchang in 2019,a total of 32QTLs related to grain shape and grain weight were detected.Among of the QTLs,six QTLs including 1000-grain weight locus qTGW2,grain length locus qGL12,grain width locus qGW2.2 and qGW5,grain width/length qLWR5,grain thickness qGT4.2.These QTLs were located in chromosome 2,12,5 and 4.(2)A total of 6 QTLs related to grain-filling rate were screened in the two different environments of the mid-season in 2019 and 2020 at Nanchang.Among of them,we screened the qGFR1.1 related to grain-filling rate on 1-5 days after anthesis,the qGFR3,qGFR5 and qGFR7.2 related to grain-filling rate on 5-10 days after antheis,the qGFR1.2and qGFR7.1 related to grain-filling rate on 10-15 days after anthesis.However,we did not screen the common QTLs under environments of Nanchang in 2019 and Nanchang in 2020.更多还原