水稻谷蛋白合成途径关键基因的图位克隆与功能研究
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摘要
水稻种子在胚乳中积累大量的储藏蛋白，约占籽粒干重的6-10％，其中谷蛋白占80％左右，贮存于PBⅡ中，任何对谷蛋白含量和组成的改变都会引起稻米品质的变化。虽然几乎所有的谷蛋白基因已经得到克隆，但人们对谷蛋白的表达调控及谷蛋白的合成、转运和沉积等过程尚缺乏足够的了解。利用突变体结合图位克隆技术是解决上述问题的捷径。我们通过对9000多份资源的筛选获得了17份谷蛋白突变体，对其中的低谷蛋白突变体W3660进行了精细定位，并将紧密连锁的分子标记应用于辅助选择育种；图位克隆了谷蛋白前体剧增突变体W379的突变基因，并对其突变的机理进行了深入的探讨；图位克隆了谷蛋白前体部分增加突变体Q4041的突变基因，并对该突变体进行了基因芯片的研究。研究结果如下：
1谷蛋白突变体的筛选
利用SDS-PAGE技术从地方品种、T-DNA插入系和辐射诱变突变体中筛选获得了17份突变体材料，突变频率仅0.189％，分为3种类型：低谷蛋白突变体，共8份，其特点是谷蛋白成熟的酸性和碱性亚基大量减少，球蛋白和醇溶蛋白含量大量增加。该类型的突变体可以用作培育适合肾脏病人和糖尿病人食用的低谷蛋白水稻品种，同时还可以用来分离谷蛋白表达调控相关基因；谷蛋白前体增加突变体(57H)，共7份，表现为谷蛋白57kD前体不同程度的增加，酸性和碱性亚基相应减少，该突变体可以用来克隆谷蛋白转运、沉积途径中的关键基因；球蛋白缺失体，共2份，该类型突变体有可能是球蛋白基因的突变，亦可能是其关键的调控基因的突变，同时聚合球蛋白缺失性状和低谷蛋白性状将有助于培育可溶性蛋白含量更低的水稻品种。
2 W3660突变基因的精细定位与分子标记辅助选择
利用粳稻品种W3660与籼稻品种惊人糯杂交构建的F_2群体及其衍生群体对控制W3660低谷蛋白性状的基因进行了研究，结果显示：突变性状受显性单基因控制。采用SSR标记技术，将该基因定位于第2染色体标记SSR2-001／SSR2-004和RM1358之间，距SSR2-001／SSR2-004和RMl358的遗传距离分别为1.1cM和3.8cM。虽然W3660的突变基因与Lgc-1位于同一个定位区间，但二者是不同的突变体。我们分析了与目的基因紧密连锁的分子标记在W3660与7个粳稻品种之间的多态性，将存在多态的SSR2-004和RM1358用于分子标记辅助选择，并计算获得两个标记在粳／粳交组合中辅助选择的效率分别为96.8％和92.7％，而采用双标记进行选择的效率则高达99.8％，从而为突变基因的分子标记辅助育种奠定了基础。
3 W379突变基因的克隆及其功能研究
利用W379／培矮64∥W379 BC_1F_1群体和W379／培矮64 F_2群体，判定W379谷蛋前体剧增性状受一对隐性核基因的控制，并将突变基因精细定位于第4染色体标记RM4-001和IN4-001间，两侧标记同位于BAC克隆OSJNBa0091D06中，中间的物理距离约30kb，利用生物信息学的方法，发现该区域存在一个液泡加工酶基因，并命名为osVpel，采用RT-PCR的方法获得了该基因的编码区，序列比对后发现，突变体和野生型间只有一个核苷酸的差异，导致了W379中osVPE1蛋白的269位由Cys突变为Gly。酶活性测定显示，W379发育胚乳中Asn特异剪切活性不到日本晴中的10％，功能互补试验证实了osVpel就是目的基因。半定量RT-PCR分析表明，osVpel的表达模式和表达水平在日本晴和W379间无显著差异，亚细胞定位结果显示突变的蛋白亦分选进入液泡中。体外表达并纯化了osVPE1片段，制备了多克隆抗体，采用粗酶提取液提取蛋白进行的Western杂交显示，W379胚乳中的osVPE1(C269G)大都以前体的形式存在，而日本晴胚乳中则存在成熟的蛋白形式；而采用总蛋白提取液提取蛋白进行的Western杂交显示，W379中的VPE成熟蛋白分子量小于日本晴中的正常蛋白，暗示osVPE1(C269G)成熟时的剪切发生了错误，导致其功能的丧失。同时，突变体和野生型中的蛋白在体外温浴时，野生型蛋白能够进行正确的自我剪切成熟，而突变体只能够进行剪切形成中间形式的蛋白，不能形成成熟的蛋白。
4 Q4041突变基因的克隆及功能研究
我们从N22辐射诱变突变体库中筛选获得了一谷蛋白前体部分增加的突变体Q4041，该突变体还表现为成熟酸性和碱性亚基的部分减少，脂肪含量、游离氨基酸含量和赖氨酸含量大大增加，直链淀粉含量下降，种子变小，胚乳不透明等。对成熟胚乳自然断面的扫描电镜观察显示，Q4041中淀粉粒形状不规则，且排列疏松。突变体和野生型正反交试验表明突变性状受单个隐性核基因控制。利用Q4041／热研2号F_2群体将目的基因定位于第12染色体末端BAC克隆OJ1584_D02中27kb的区域，利用生物信息学方法预测到该区域存在一个Rab5a基因，我们将它命名为osRab5a。基因组和cDNA序列分析表明osrab5a在第3个外元缺失了13bp，造成移码突变，蛋白翻译提前终止。我们构建了一系列的转基因载体对该基因进行功能互补研究、亚细胞定位、体外表达研究等。同时我们对Q4041和N22发育胚乳RNA进行了基因芯片的研究，发现了一大批表达发生显著变化的基因(Foldchange＞2或Foldchange＜0.5为界)，包括1个表达下调的基因(osRab5a)和1000多个表达上调的基因，表达上调的基因中包括糖代谢和淀粉合成相关基因，Ca~(2+)结合相关基因(calmodulin，EF-hand protein)，过氧化物酶基因，真核生物转录因子等，为全面阐述osRab5a的功能奠定了基础。
Rice (Oryza sativa L.) seeds accumulate large amounts of storage proteins (seed storage proteins, SSP) in endosperm during seed development. Glutelins account for about 80% of the total proteins and deposite in protein body II (PBII). Any change in glutelin content or composition will alter rice quality. Although most of the glutelin genes or cDNAs had been cloned, the expression and regulation of glutelin genes and the cellular processes underlying glutelin biosynthesis, transport and deposition remain to be understood. The development and utilization of mutants combining the strategy of map-based cloning is one efficient approach in resolving above problems. To obtain mutants, over 9,000 lines including landraces, T-DNA insertion lines and radiation induced mutation lines were subjected to SDS-PAGE analysis. Totally we obtained 17 mutants, of which 3 ones have been studied extensively. To the low glutelin content mutant, W3660, we fine-mapped the mutated gene, and applied the flanking markers in marker-assisted selection. To the 57H mutant, W379, we isolated the mutated gene through map-based cloning strategy and elucidated the molecular mechamism underlying glutelin precursors accumulation in W379. To another 57H mutant, Q4041, we also map-based isolated the mutated gene, and a microarray experiment was done to search for the reason why the 57kD precursor was partly accumulated and to explore the affected pathway(s) caused by the mutation. The results are as followings:
1. Screening of Seed Storage Protein mutants
After screening over 9000 lines, 17 SSP mutants were obtained with a mutation fequency of only 0.189%. These mutants can be simply classified into 3 types: a, the LGC mutant which is characterized by low content of glutelins and high content of prolamins and globulin. It can be used as not only the donor parent in developing a rice cultivar suitable for patients with diabetes and kidney disease, but fairly good materials for isolation genes responsible for the expression and regulation of glutelin genes as well; b, the 57H mutant which is characterized by high amount of 57kD polypeptides and low amounts of mature glutelin subunits. The seven 57H mutants are excellent materials for isolating key genes involved in the regulation of transport and deposition of glutelins; c, the globulin deletion (gd) mutant which is characterized by the missing of globulin polypeptide. This may be caused by the mutation of globulin itself or gene(s) regulating the globulin biosynthesis. Moreover, pyramiding genes of Lgc and gd will develop a cultivar with even lower amount of soluble proteins.
2. Fine mapping and marker-assisted selection of a low glutelin content gene in W3660
Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. W3660 is a novel mutant characterized by low glutelin content. F_2 and F_3 populations were derived from a cross between W3660 and Jingrennuo to map the gene(s) responsible for the low glutelin content. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The distances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. Although the chromosome position of this gene was similar to LGC-1, they are different genes. SSR profiles of seven japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker-assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively, and thus these markers will be suitable for the selection of low glutelin content rice.
3. Isolation and characterization of a vacuolar processing enzyme responsible for the maturation of glutelins in rice
Rice accumulates prolamins and glutelins as storage protein. The latter storage proteins are synthesized on rough endoplasmic reticulum (rER) as 57kD precursors and are sorted into protein storage vacuoles, where they are processed into acidic and basic subunits forms. We report here a rice 57H mutant, W379, which abnormally accumulates glutelin precursors. Genetic analysis revealed that the phenotype in W379 was controlled by a single recessive nuclear gene. Through map-based cloning, a homolog of the arabidopsisβVpe was isolated, which was tentatively nominated osVpe1. The coding sequence of osVpe1 has 1494 base pairs, and encodes a protein of 494 amino acids. Sequence analysis showed that only one nucleotide was different between W379 and Nipponbare, resulting in a change of Cys269 to Gly. The expression pattern and expression level of osVpe1 in developing endosperm were very similar in the two cultivars. Subcellular localization indicated the deformed protein was also sorted into vacuoles as WT protein did. But the enzyme activity was almost lost in W379. Western blotting revealed that osVpe1 was cleaved by mistake, resulting in a smaller size of mature protein. Our findings suggest that osVpe1 plays a crucial role in the maturation of rice glutelins, and the Cys269 residue is a key residue in maintaining its' Asn-specific cleavage activity.
4. Isolation and Characterization a small GTP binding protein, osRab5a, involved in the transport of storage proteins in rice
A new 57H mutant named Q4041 was obtained from radiation induced mutation lines, Q4041 abnormally accumulated glutelin precursors, but its' amount was less than W379. The seed of Q4041 was smaller and had opaque endosperm. This indicated a defect in starch biosynthesis. SEM analysis of the mature endosperm showed that the opaque endosperm comprised round and loosely packed, compound starch granules. Genetic analysis showed the mutant trait in Q4041 was controlled by a single recessive nuclear gene, the 57H trait and opaque trait were controlled by the same gene. An F_2 population derived from the cross between Q4041 and Reyan2 was adopted to map the gene responsible for the 57H trait. Using PCR-based molecular markers, we mapped the gene on Chromosome 12, in the region between 12-170 and 12-191, with a physical distance of 27kb. In this region, three genes were predicted, including osRab5a, ubiquitin E3 ligase gene and a retrotransponson gene. Sequence analysis showed no differece in E3 ligase gene between WT and mutant. Semiquantitative RT-PCR analysis showed that the expression of osRab5a in Q4041 was much lower than in WT. Further we sequeced the genomic DNA and cDNA of osRab5a in Q4041 and found a 13bp deletion in the third exon. Thus we obtained the gene responsible for the 57H trait in Q4041. To explore the affected pathway(s) caused by this mutation, a microarray experiment was done, and found that totally only one gene was downregulated and over 1000 genes was upredulated in Q4041 compared with WT(Foldchange>2 or foldchange<0.5, and P<0.05), including osRab5a itself, sugar metabolism and starch biosynthesis related genes, Calcium binding proteins, peroxidase gene family, Heat shock proteins, and Eukaryotic transcription initiation factor, etc. This provides a basis for the characterization of osRab5a.
1谷蛋白突变体的筛选
利用SDS-PAGE技术从地方品种、T-DNA插入系和辐射诱变突变体中筛选获得了17份突变体材料，突变频率仅0.189％，分为3种类型：低谷蛋白突变体，共8份，其特点是谷蛋白成熟的酸性和碱性亚基大量减少，球蛋白和醇溶蛋白含量大量增加。该类型的突变体可以用作培育适合肾脏病人和糖尿病人食用的低谷蛋白水稻品种，同时还可以用来分离谷蛋白表达调控相关基因；谷蛋白前体增加突变体(57H)，共7份，表现为谷蛋白57kD前体不同程度的增加，酸性和碱性亚基相应减少，该突变体可以用来克隆谷蛋白转运、沉积途径中的关键基因；球蛋白缺失体，共2份，该类型突变体有可能是球蛋白基因的突变，亦可能是其关键的调控基因的突变，同时聚合球蛋白缺失性状和低谷蛋白性状将有助于培育可溶性蛋白含量更低的水稻品种。
2 W3660突变基因的精细定位与分子标记辅助选择
利用粳稻品种W3660与籼稻品种惊人糯杂交构建的F_2群体及其衍生群体对控制W3660低谷蛋白性状的基因进行了研究，结果显示：突变性状受显性单基因控制。采用SSR标记技术，将该基因定位于第2染色体标记SSR2-001／SSR2-004和RM1358之间，距SSR2-001／SSR2-004和RMl358的遗传距离分别为1.1cM和3.8cM。虽然W3660的突变基因与Lgc-1位于同一个定位区间，但二者是不同的突变体。我们分析了与目的基因紧密连锁的分子标记在W3660与7个粳稻品种之间的多态性，将存在多态的SSR2-004和RM1358用于分子标记辅助选择，并计算获得两个标记在粳／粳交组合中辅助选择的效率分别为96.8％和92.7％，而采用双标记进行选择的效率则高达99.8％，从而为突变基因的分子标记辅助育种奠定了基础。
3 W379突变基因的克隆及其功能研究
利用W379／培矮64∥W379 BC_1F_1群体和W379／培矮64 F_2群体，判定W379谷蛋前体剧增性状受一对隐性核基因的控制，并将突变基因精细定位于第4染色体标记RM4-001和IN4-001间，两侧标记同位于BAC克隆OSJNBa0091D06中，中间的物理距离约30kb，利用生物信息学的方法，发现该区域存在一个液泡加工酶基因，并命名为osVpel，采用RT-PCR的方法获得了该基因的编码区，序列比对后发现，突变体和野生型间只有一个核苷酸的差异，导致了W379中osVPE1蛋白的269位由Cys突变为Gly。酶活性测定显示，W379发育胚乳中Asn特异剪切活性不到日本晴中的10％，功能互补试验证实了osVpel就是目的基因。半定量RT-PCR分析表明，osVpel的表达模式和表达水平在日本晴和W379间无显著差异，亚细胞定位结果显示突变的蛋白亦分选进入液泡中。体外表达并纯化了osVPE1片段，制备了多克隆抗体，采用粗酶提取液提取蛋白进行的Western杂交显示，W379胚乳中的osVPE1(C269G)大都以前体的形式存在，而日本晴胚乳中则存在成熟的蛋白形式；而采用总蛋白提取液提取蛋白进行的Western杂交显示，W379中的VPE成熟蛋白分子量小于日本晴中的正常蛋白，暗示osVPE1(C269G)成熟时的剪切发生了错误，导致其功能的丧失。同时，突变体和野生型中的蛋白在体外温浴时，野生型蛋白能够进行正确的自我剪切成熟，而突变体只能够进行剪切形成中间形式的蛋白，不能形成成熟的蛋白。
4 Q4041突变基因的克隆及功能研究
我们从N22辐射诱变突变体库中筛选获得了一谷蛋白前体部分增加的突变体Q4041，该突变体还表现为成熟酸性和碱性亚基的部分减少，脂肪含量、游离氨基酸含量和赖氨酸含量大大增加，直链淀粉含量下降，种子变小，胚乳不透明等。对成熟胚乳自然断面的扫描电镜观察显示，Q4041中淀粉粒形状不规则，且排列疏松。突变体和野生型正反交试验表明突变性状受单个隐性核基因控制。利用Q4041／热研2号F_2群体将目的基因定位于第12染色体末端BAC克隆OJ1584_D02中27kb的区域，利用生物信息学方法预测到该区域存在一个Rab5a基因，我们将它命名为osRab5a。基因组和cDNA序列分析表明osrab5a在第3个外元缺失了13bp，造成移码突变，蛋白翻译提前终止。我们构建了一系列的转基因载体对该基因进行功能互补研究、亚细胞定位、体外表达研究等。同时我们对Q4041和N22发育胚乳RNA进行了基因芯片的研究，发现了一大批表达发生显著变化的基因(Foldchange＞2或Foldchange＜0.5为界)，包括1个表达下调的基因(osRab5a)和1000多个表达上调的基因，表达上调的基因中包括糖代谢和淀粉合成相关基因，Ca~(2+)结合相关基因(calmodulin，EF-hand protein)，过氧化物酶基因，真核生物转录因子等，为全面阐述osRab5a的功能奠定了基础。
Rice (Oryza sativa L.) seeds accumulate large amounts of storage proteins (seed storage proteins, SSP) in endosperm during seed development. Glutelins account for about 80% of the total proteins and deposite in protein body II (PBII). Any change in glutelin content or composition will alter rice quality. Although most of the glutelin genes or cDNAs had been cloned, the expression and regulation of glutelin genes and the cellular processes underlying glutelin biosynthesis, transport and deposition remain to be understood. The development and utilization of mutants combining the strategy of map-based cloning is one efficient approach in resolving above problems. To obtain mutants, over 9,000 lines including landraces, T-DNA insertion lines and radiation induced mutation lines were subjected to SDS-PAGE analysis. Totally we obtained 17 mutants, of which 3 ones have been studied extensively. To the low glutelin content mutant, W3660, we fine-mapped the mutated gene, and applied the flanking markers in marker-assisted selection. To the 57H mutant, W379, we isolated the mutated gene through map-based cloning strategy and elucidated the molecular mechamism underlying glutelin precursors accumulation in W379. To another 57H mutant, Q4041, we also map-based isolated the mutated gene, and a microarray experiment was done to search for the reason why the 57kD precursor was partly accumulated and to explore the affected pathway(s) caused by the mutation. The results are as followings:
1. Screening of Seed Storage Protein mutants
After screening over 9000 lines, 17 SSP mutants were obtained with a mutation fequency of only 0.189%. These mutants can be simply classified into 3 types: a, the LGC mutant which is characterized by low content of glutelins and high content of prolamins and globulin. It can be used as not only the donor parent in developing a rice cultivar suitable for patients with diabetes and kidney disease, but fairly good materials for isolation genes responsible for the expression and regulation of glutelin genes as well; b, the 57H mutant which is characterized by high amount of 57kD polypeptides and low amounts of mature glutelin subunits. The seven 57H mutants are excellent materials for isolating key genes involved in the regulation of transport and deposition of glutelins; c, the globulin deletion (gd) mutant which is characterized by the missing of globulin polypeptide. This may be caused by the mutation of globulin itself or gene(s) regulating the globulin biosynthesis. Moreover, pyramiding genes of Lgc and gd will develop a cultivar with even lower amount of soluble proteins.
2. Fine mapping and marker-assisted selection of a low glutelin content gene in W3660
Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. W3660 is a novel mutant characterized by low glutelin content. F_2 and F_3 populations were derived from a cross between W3660 and Jingrennuo to map the gene(s) responsible for the low glutelin content. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The distances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. Although the chromosome position of this gene was similar to LGC-1, they are different genes. SSR profiles of seven japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker-assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively, and thus these markers will be suitable for the selection of low glutelin content rice.
3. Isolation and characterization of a vacuolar processing enzyme responsible for the maturation of glutelins in rice
Rice accumulates prolamins and glutelins as storage protein. The latter storage proteins are synthesized on rough endoplasmic reticulum (rER) as 57kD precursors and are sorted into protein storage vacuoles, where they are processed into acidic and basic subunits forms. We report here a rice 57H mutant, W379, which abnormally accumulates glutelin precursors. Genetic analysis revealed that the phenotype in W379 was controlled by a single recessive nuclear gene. Through map-based cloning, a homolog of the arabidopsisβVpe was isolated, which was tentatively nominated osVpe1. The coding sequence of osVpe1 has 1494 base pairs, and encodes a protein of 494 amino acids. Sequence analysis showed that only one nucleotide was different between W379 and Nipponbare, resulting in a change of Cys269 to Gly. The expression pattern and expression level of osVpe1 in developing endosperm were very similar in the two cultivars. Subcellular localization indicated the deformed protein was also sorted into vacuoles as WT protein did. But the enzyme activity was almost lost in W379. Western blotting revealed that osVpe1 was cleaved by mistake, resulting in a smaller size of mature protein. Our findings suggest that osVpe1 plays a crucial role in the maturation of rice glutelins, and the Cys269 residue is a key residue in maintaining its' Asn-specific cleavage activity.
4. Isolation and Characterization a small GTP binding protein, osRab5a, involved in the transport of storage proteins in rice
A new 57H mutant named Q4041 was obtained from radiation induced mutation lines, Q4041 abnormally accumulated glutelin precursors, but its' amount was less than W379. The seed of Q4041 was smaller and had opaque endosperm. This indicated a defect in starch biosynthesis. SEM analysis of the mature endosperm showed that the opaque endosperm comprised round and loosely packed, compound starch granules. Genetic analysis showed the mutant trait in Q4041 was controlled by a single recessive nuclear gene, the 57H trait and opaque trait were controlled by the same gene. An F_2 population derived from the cross between Q4041 and Reyan2 was adopted to map the gene responsible for the 57H trait. Using PCR-based molecular markers, we mapped the gene on Chromosome 12, in the region between 12-170 and 12-191, with a physical distance of 27kb. In this region, three genes were predicted, including osRab5a, ubiquitin E3 ligase gene and a retrotransponson gene. Sequence analysis showed no differece in E3 ligase gene between WT and mutant. Semiquantitative RT-PCR analysis showed that the expression of osRab5a in Q4041 was much lower than in WT. Further we sequeced the genomic DNA and cDNA of osRab5a in Q4041 and found a 13bp deletion in the third exon. Thus we obtained the gene responsible for the 57H trait in Q4041. To explore the affected pathway(s) caused by this mutation, a microarray experiment was done, and found that totally only one gene was downregulated and over 1000 genes was upredulated in Q4041 compared with WT(Foldchange>2 or foldchange<0.5, and P<0.05), including osRab5a itself, sugar metabolism and starch biosynthesis related genes, Calcium binding proteins, peroxidase gene family, Heat shock proteins, and Eukaryotic transcription initiation factor, etc. This provides a basis for the characterization of osRab5a.
引文
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