香蕉果实可溶性淀粉合成酶基因MaSSⅢ-1的分子特征与时空表达模式
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摘要
为弄清香蕉果实可溶性淀粉合成酶基因(MaSSⅢ-1)的分子特征及时空表达模式,以‘巴西蕉’果肉为试材,采用PCR法进行MaSSⅢ-1基因克隆,通过Quantitative real-time PCR (qPCR)和Western blot技术对MaSSⅢ-1基因及其蛋白表达模式进行分析。结果显示:MaSSⅢ-1基因cDNA全长为2397 bp,编码798个氨基酸,包括4个典型的结构域,GenBank登录号为KU757067。MaSSⅢ-1基因在香蕉根、球茎、花和苞片中表达量较低,在叶、果皮和果肉中表达量较高;随着香蕉果实发育,MaSSⅢ-1基因表达量逐渐上升,在抽蕾后50～60天达到最大;随着果实采后成熟,MaSSⅢ-1表达量在采后5天达到最大,随后逐渐下降。在香蕉果实不同发育及采后成熟阶段,MaSSⅢ-1蛋白呈现出与mRNA水平相一致的表达趋势。证明MaSSⅢ-1基因的表达不仅涉及到香蕉果实支链淀粉合成代谢,可能还涉及采后早期果实成熟或者支链淀粉降解。
To clarify the molecular characteristics and temporal and spatial expression patterns of soluble starch synthase Ⅲ-1 gene(MaSSⅢ-1), using the‘Brazilian'banana pulps as the test material, the MaSSⅢ-1 genewas cloned by PCR method. Quantitative real-time PCR(qPCR) and Western blot technology were used toanalyze MaSSⅢ-1 gene and its protein expression pattern. The results showed that the cDNA full-length of MaSSⅢ-1 gene(accession No. KU757067) was 2397 bp encoding 798 amino acids with four typical domains.The expression level of MaSSⅢ-1 was low in roots, bulbs, flowers, and bracts, but it exhibited high expressionin leaves, peels, and pulps. Moreover, MaSSⅢ-1 expression increased gradually with banana fruitdevelopment, and then reached the highest expression in 50-60 d after emergence from the pseudostem. The expression level of MaSSⅢ-1 reached the maximum value in 5 d after harvest and then decreased graduallyduring postharvest ripening in banana fruit. The expression pattern of MaSSⅢ-1 protein was consistent withthe expression trend at mRNA level during banana fruit development and ripening. These results suggestedthat the expression of MaSSⅢ-1 gene may involve not only amylopectin biosynthesis but also early fruitripening or amylopectin degradation in banana.
To clarify the molecular characteristics and temporal and spatial expression patterns of soluble starch synthase Ⅲ-1 gene(MaSSⅢ-1), using the‘Brazilian'banana pulps as the test material, the MaSSⅢ-1 genewas cloned by PCR method. Quantitative real-time PCR(qPCR) and Western blot technology were used toanalyze MaSSⅢ-1 gene and its protein expression pattern. The results showed that the cDNA full-length of MaSSⅢ-1 gene(accession No. KU757067) was 2397 bp encoding 798 amino acids with four typical domains.The expression level of MaSSⅢ-1 was low in roots, bulbs, flowers, and bracts, but it exhibited high expressionin leaves, peels, and pulps. Moreover, MaSSⅢ-1 expression increased gradually with banana fruitdevelopment, and then reached the highest expression in 50-60 d after emergence from the pseudostem. The expression level of MaSSⅢ-1 reached the maximum value in 5 d after harvest and then decreased graduallyduring postharvest ripening in banana fruit. The expression pattern of MaSSⅢ-1 protein was consistent withthe expression trend at mRNA level during banana fruit development and ripening. These results suggestedthat the expression of MaSSⅢ-1 gene may involve not only amylopectin biosynthesis but also early fruitripening or amylopectin degradation in banana.
引文
[1]苗红霞,金志强,刘伟鑫,等.香蕉果实抗性淀粉变化及其与其他类型淀粉相关性分析[J].中国农业科学,2013,46(24):5180-5187.
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[11]苗红霞,金志强,孙佩光,等.香蕉果实MaGBSSI-3基因家族的克隆及表达分析[J].北方园艺,2014(20):115-120.
[12] Livak K J, Schimittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTMethod[J].Methods,2001,25(4):402-408.
[13] Nishi A, Nakamura Y, Tanaka N, et al. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm[J]. Plant Physiology,2001,127:459-472.
[14] Yang Z F, Wang Y F, Xu S H, et al. Molecular evolution and functional divergence of soluble starch synthase genes in cassava(Manihot Esculenta Crantz)[J]. Evolutionary Bioinformatics,2013,9:239-240.
[15]张莉,印荔,杨见秋,等.莲藕可溶性淀粉合成酶基因LrSSS的克隆与表达特性分析[J].园艺学报,2015,42(3):496-504.
[16] Szydlowski N, Ragel P, Raynaud S, et al. Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthases[J]. Plant Cell,2009,21:2443-2457.
[17] Zhang X, Szydlowski N, DelvalléD, et al. Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis[J]. BMC Plant Biology,2008,8:96.
[18] Barchiesi J, Hedin N, Iglesias A A, et al. Identification of a novel starch synthase II from the picoalgae Ostreococcus tauri[J].Biochimie,2017,133:37-44.
[19] Zhu F, Bertoft E, Seetharaman K. Distribution of branches in whole starches from maize mutants deficient in starch synthase III[J].Journal of Agricultural&Food Chemistry,2014,62:4577-4583.
[20] Li Z, Mouille G, Kosar-Hashemi B, et al. The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family[J]. Plant Physiology,2000,123:613-624.
[2] Miura S, Crofts N, Saito Y, et al. Starch synthase IIa-deficient mutant rice line produces endosperm starch with lower gelatinization temperature than japonica rice cultivars[J]. Frontier in Plant Science,2018,9:645.
[3] Dian W, Jiang H W, Wu P. Evolution and expression analysis of starch synthase III and IV in rice[J]. Journal of Experimental Botany,2005,56:623-632.
[4] Cao H, James M G, Myers A M. Purification and characterization of soluble starch synthases from maize endosperm[J]. Archives of Biochemistry and Biophysics,2000,373(1):135-146.
[5] Fujita N, Yoshida M, Kondo T, et al. Characterization of SSIIIadeficient mutants of rice:the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm[J]. Plant Physiology,2007,144(4):2009-2023.
[6] Roldán I, Wattebled F, Mercedes Lucas M, et al. The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation[J]. Plant Journal,2007,49(3):492-504.
[7] Marshall J, Sidebottom C, Debet M, et al. Identification of the major starch synthase in the soluble fraction of potato tubers[J].Plant Cell,1996,8(7):1121-1135.
[8] Kossmann J, Abel G J, Springer F, et al. Cloning and functional analysis of a cDNA encoding a starch synthase from potato(Solanum tuberosum L.)that is predominantly expressed in leaf tissue[J]. Planta,1999,208(4):503-511.
[9] Hirose T, Terao T. A comprehensive expression analysis of the starch synthase gene family in rice(Oryza sativa L.)[J]. Planta,2004,220(1):9-16.
[10]罗明,肖月华,侯磊,等.棉花LM结构域基因(GhLM1)的克隆与表达分析[J].遗传学报,2003,30(2):175-182.
[11]苗红霞,金志强,孙佩光,等.香蕉果实MaGBSSI-3基因家族的克隆及表达分析[J].北方园艺,2014(20):115-120.
[12] Livak K J, Schimittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTMethod[J].Methods,2001,25(4):402-408.
[13] Nishi A, Nakamura Y, Tanaka N, et al. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm[J]. Plant Physiology,2001,127:459-472.
[14] Yang Z F, Wang Y F, Xu S H, et al. Molecular evolution and functional divergence of soluble starch synthase genes in cassava(Manihot Esculenta Crantz)[J]. Evolutionary Bioinformatics,2013,9:239-240.
[15]张莉,印荔,杨见秋,等.莲藕可溶性淀粉合成酶基因LrSSS的克隆与表达特性分析[J].园艺学报,2015,42(3):496-504.
[16] Szydlowski N, Ragel P, Raynaud S, et al. Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthases[J]. Plant Cell,2009,21:2443-2457.
[17] Zhang X, Szydlowski N, DelvalléD, et al. Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis[J]. BMC Plant Biology,2008,8:96.
[18] Barchiesi J, Hedin N, Iglesias A A, et al. Identification of a novel starch synthase II from the picoalgae Ostreococcus tauri[J].Biochimie,2017,133:37-44.
[19] Zhu F, Bertoft E, Seetharaman K. Distribution of branches in whole starches from maize mutants deficient in starch synthase III[J].Journal of Agricultural&Food Chemistry,2014,62:4577-4583.
[20] Li Z, Mouille G, Kosar-Hashemi B, et al. The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family[J]. Plant Physiology,2000,123:613-624.