大豆GmDFR基因克隆及其抵御缺铁胁迫功能的鉴定  

Cloning of GmDFR gene from Soybean(Glycine max) and Identification of Its Function on Resistance to Iron Deficiency

作　　者：石卓 李洪 高铭 郭长虹[1] 郭东林[1] 毕影东[2] SHI Zhuo;LI Hong;GAO Ming;GUO Chang-Hong;GUO Dong-Lin;BI Ying-Dong(College of Life Science and Technology/Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province,Harbin Normal University,Harbin 150025,China;Tillage and Cultivation Institute,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院/黑龙江省分子细胞遗传与遗传育种重点实验室,哈尔滨150025 [2]黑龙江省农业科学院耕作栽培研究所,哈尔滨150086

出　　处：《农业生物技术学报》2023年第2期259-272,共14页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972507);国家自然科学基金区域联合基金(U21A20182);黑龙江省杰出青年基金(JQ2019C003);赛信生物科技有限公司科技开发项目(202101)。

摘　　要：二氢黄酮醇-4-还原酶(dihydroflavonol-4-reductase, DFR)在植物的花青素合成中发挥关键催化作用。花青素能保护组织免受非生物胁迫诱发的氧化应激,也可以作为螯合剂促进植物对铁的吸收。本研究克隆了大豆(Glycine max) GmDFR基因(GenBank No. MN547961) CDS及其启动子片段pGmDFR(GenBank No. MW455109),并对其生物信息学和抵御缺铁胁迫的功能进行了分析。结果显示,GmDFR的CDS全长1 020 bp,编码339个氨基酸。GmDFR具有典型的PLN02650和WcaG超家族结构域,属于DFR和核苷二磷酸糖异构酶家族,GmDFR蛋白不是跨膜蛋白。系统发育分析表明,大豆GmDFR与野生大豆(G. soja) GsDFR的相似度最高,为99.12%。半定量RT-PCR检测表明GmDFR在大豆各组织中均有表达,且在根中表达水平最高。克隆获得启动子片段pGmDFR,含有多个激素和逆境胁迫响应元件。缺铁(0mmol/L Fe-EDTA)胁迫下转GmDFR基因烟草(Nicotiana tabacum)的花青素含量、叶绿素含量显著高于野生型(P<0.05);丙二醛及过氧化氢、超氧阴离子含量显著低于野生型(P<0.05);而抗氧化酶(SOD, POD和CAT)活性显著高于野生型(P<0.05),表明转GmDFR烟草具有一定的抵御缺铁胁迫的能力。本研究为GmDFR在大豆抵抗缺铁胁迫及提高抗氧化能力中的应用提供科学依据,也为大豆分子育种提供新的基因资源。Dihydroflavonol-4-reductase(DFR) plays a key catalytic role in anthocyanin synthesis.Anthocyanin can protect tissues from oxidative damage induced by abiotic stress, and can also be used as chelating agents to promote plant iron absorption. In this study, GmDFR gene(GenBank No. MN547961) and its promoter p GmDFR fragment(GenBank No. MW455109) were cloned from soybean(Glycine max). The CDS length of GmDFR gene was 1 020 bp and encoded 339 amino acids. GmDFR had typical PLN02650 and WcaG superfamily domains, belongs to dihydroflavonol 4-reductase and nucleoside diphosphate sugar isomerase family, GmDFR protein was not a transmembrane protein. Phylogenetic analysis showed that soybean GmDFR was most closely related to wild soybean(G. soja) GsDFR, with a similarity of 99.12%.Semi-quantitative RT-PCR detection showed that GmDFR was expressed in soybean tissues and the highest expression level was in roots. The pGmDFR promoter fragment was obtained by cloning, which contained multiple hormone and stress response elements. Under iron deficiency, the anthocyanin content and chlorophyll content of GmDFR transgenic tobacco(Nicotiana tabacum) were significantly higher than that of WT(P<0.05);the contents of malondialdehyde, hydrogen peroxide and superoxide anion were significantly lower than that of WT(P<0.05);and the activities of antioxidant enzymes(SOD, POD and CAT) were significantly higher than that of WT(P<0.05). These results indicated that transgenic GmDFR tobacco had certain ability to resist iron deficiency. This study provides scientific basis for the application of GmDFR gene in soybean resistance to iron deficiency, and also provides new gene resources for soybean molecular breeding.

关 键 词：大豆 二氢黄酮醇-4-还原酶(DFR) 表达分析 生物信息学分析 功能分析 

分 类 号：S565.1[农业科学—作物学]