枣<i>ZjIAA9</i>生物信息学分析及功能验证

doi:10.7668/hbnxb.20195979

摘要/Abstract

摘要： Aux/IAA基因家族成员编码的蛋白通过调节生长素(IAA)信号转导途径调控植物的胚胎形成、种子发育等多种发育过程。为探究Aux/IAA基因家族在枣胚发育过程中的作用,利用胚高度可育枣品种灰枣和胚低度可育枣品种孔府酥脆枣的转录组数据,对影响枣胚发育过程的Aux/IAA基因家族的关键基因进行生物信息学分析和功能验证。结果表明,经过基因克隆获得了一个长度为1 731 bp的Aux/IAA基因家族的基因序列,编码362个氨基酸。蛋白质结构预测显示,其二级结构以无规则卷曲为主,且具有典型的IAA9蛋白保守域,将其命名为ZjIAA9。同源性分析结果显示,ZjIAA9与红栎中的RGQ29_009000、栓皮栎中的CFP56_014209亲缘关系较近。通过农杆菌介导的叶盘法转基因获得了Micro-Tom小番茄转基因植株。对Micro-Tom小番茄转基因植株的生长素含量进行测定后发现,转基因植株果肉的IAA含量高于未转基因植株,表明ZjIAA9可能参与生长素的生物合成。与野生型相比,转基因植株的表现为无籽或少籽状,表明枣ZjIAA9可能参与调控枣胚胎发育过程。

关键词: 枣, 胚发育, ZjIAA9, 生物信息学分析, 功能验证

Abstract:

The members of the Aux/IAA gene family encode proteins that regulate various developmental processes in plants,such as embryogenesis and seed development,by modulating the auxin (IAA) signal transduction pathway.To explore the role of Aux/IAA family genes in jujube embryo development,this study performed bioinformatics analysis and functional verification on the key genes of the Aux/IAA family that affect jujube embryo development,using transcriptome data of Huizao (a jujube variety with high embryo fertility) and Kongfusucui (a jujube variety with low embryo fertility).Results showed that an Aux/IAA family gene sequence with a length of 1 731 bp was obtained via gene cloning,which encoded 362 amino acids.Protein structure prediction indicated that its secondary structure was mainly composed of random coil and that it had a typical conserved domain of the IAA9 protein;thus,it was named ZjIAA9.Homology analysis revealed that ZjIAA9 was closely related to RGQ29_009000 in Quercus rubra and CFP56_014209 in Quercus suber.Transgenic Micro-Tom tomato plants were obtained via Agrobacterium-mediated leaf disc transformation.Determination of auxin content in transgenic Micro-Tom tomato plants showed that the IAA content in the pulp of transgenic plants was higher than that in non-transgenic plants,indicating that ZjIAA9 may regulate auxin biosynthesis.Compared with wild-type plants,transgenic plants showed a seedless or few-seeded phenotype,suggesting that jujube ZjIAA9 may be involved in regulating jujube embryo development.

Key words: Jujube, Embryo development, ZjIAA9, Bioinformatics analysis, Functional verification

中图分类号:

S665.1

杨梦帆, 马晴, 任海燕, 王永康, 赵爱玲, 薛晓芳, 苏万龙, 石美娟, 刘丽, 李毅. 枣ZjIAA9生物信息学分析及功能验证[J]. 华北农学报, 2025, 40(6): 77-84. doi: 10.7668/hbnxb.20195979.

YANG Mengfan, MA Qing, REN Haiyan, WANG Yongkang, ZHAO Ailing, XUE Xiaofang, SU Wanlong, SHI Meijuan, LIU Li, LI Yi. Bioinformatics Analysis and Functional Verification of ZjIAA9 in Ziziphus jujuba Mill.[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 77-84. doi: 10.7668/hbnxb.20195979.

http://www.hbnxb.net/CN/Y2025/V40/I6/77

图/表 10

图1 RNA质量检测 1—2.灰枣胚珠RNA;3—4.孔府酥脆枣胚珠RNA。

Fig.1 RNA quality analysis 1—2.Embryo sac RNA of Huizao;3—4.Embryo sac RNA of Kongfusucui.

图2 枣胚中ZjIAA9的表达量不同小写字母表示不同处理间差异显著(P<0.05)。图9同。

Fig.2 The expression level of ZjIAA9 in jujube embryos Different lowercase letters indicated significant differences between different treatments (P<0.05).The same as Fig.9.

图3 ZjIAA9基因PCR扩增产物的凝胶电泳检测 M.DNA 分子质量标准;1.ZjIAA9基因PCR扩增产物。

Fig.3 The PCR product of the ZjIAA9 gene was detected by gel electrophoresis M.DNA Marker;1.PCR amplification product of ZjIAA9.

图4 ZjIAA9的生物信息学分析 A.ZjIAA9的疏水性和亲水性分析;B.ZjIAA9蛋白结构域预测;C.ZjIAA9二级结构预测;D.ZjIAA9三级结构预测。

Fig.4 Bioinformatics analysis of ZjIAA9 A.Analysis of the hydrophobicity and hydrophilicity of the ZjIAA9 protein;B.Prediction of the structural domains of the ZjIAA9 protein; C.Prediction of the secondary structure of the ZjIAA9 protein;D.Prediction of the tertiary structure of the ZjIAA9 protein.

图5 枣ZjIAA9与其他物种中同源蛋白的序列比对分析

Fig.5 Multiple sequence alignment analysis of ZJIAA9 with its homologs from other species

图6 Micro-Tom小番茄转化流程 A.播种;B.小番茄叶片诱导产生愈伤组织和不定芽;C.不定芽潮霉素抗性筛选;D.诱导生根。

Fig.6 Flow diagram of the transformation process of Micro-Tom cherry tomatoes A.Sowing;B.Induction of callus and adventitious buds from tomato leaves;C.Hygromycin resistance screening of adventitious buds;D.Induction of rooting.

图7 转基因植株的PCR鉴定 M.2 000 bp DNA 分子质量标准;1—11.转基因植株;B.空白对照;N.阴性对照;P.阳性对照。

Fig.7 PCR identification of transgenic plants M.2 000 bp DNA Marker;1—11.Transgenic plants; B.Blank control;N.Negative control;P.Positive control.

图8 ZjIAA9对小番茄种仁发育的影响

Fig.8 The influence of ZjIAA9 on the development of the seed kernels of transgenic cherry tomatoes

图9 转基因小番茄与未转基因小番茄仁果比分析

Fig.9 Analysis of the seed-to-fruit ratio between transgenic and non-transgenic cherry tomatoes

图10 未转基因和转基因小番茄的IAA含量和基因相对表达量分析 IR.对照组种仁;N.对照组果肉;1—10.转ZjIAA9基因阳性植株果肉。大写字母用于比较IR组与其他各组,小写字母用于比较N-10组,不同字母表示差异显著(P<0.05)。

Fig.10 Comparison of IAA contents and relative gene expression levels between non-transgenic and transgenic cherry tomatoes IR.Control group kernel;N.Control group pulp;1—10.Pulp from ZjIAA9-transformed positive plants.Uppercase letters represent comparisons between the IR group and other groups; lowercase letters represent comparisons involving the N-10 group. Different letters indicate significant differences(P<0.05).

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枣ZjIAA9生物信息学分析及功能验证

Bioinformatics Analysis and Functional Verification of ZjIAA9 in Ziziphus jujuba Mill.

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