榛子全基因组ChSEP基因鉴定、多克隆抗体制备及其在胚珠中的表达研究  

Identification of ChSEP genes in the whole genome of hazelnut,preparation of polyclonal antibody and expression in ovules

作　　者：赵棣 胡献文 魏珩 程云清[1] 刘剑锋[1] Zhao Di;Hu Xianwen;Wei Heng;Cheng Yunqing;Liu Jianfeng(Jilin Provincial Key Laboratory of Plant Resource Science and Green Production,Jilin Normal University,Siping 136000,Jilin,China)

机构地区：[1]吉林师范大学吉林省植物资源科学与绿色生产重点实验室,吉林四平136000

出　　处：《北京林业大学学报》2023年第8期123-131,共9页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(32171840);吉林省科技厅中青年科技创新创业卓越人才(团队)项目(20210509033RQ)。

摘　　要：【目的】正常的胚珠充实是榛子种仁形成的前提与基础,胚珠充实不足可频繁导致瘪仁、空壳果实的形成,SEP基因被认为是可以影响胚珠发育的E类MADS-box基因。为研究SEP在榛子种仁充实过程中的重要调节作用,拟鉴定参与榛子胚珠发育的重要作用基因ChSEP,并制备ChSEP的多克隆抗体,开展正常发育与败育胚珠中ChSEP的免疫组织化学分析,以期为深入解析SEP基因调控榛子胚珠发育机制提供科学依据。【方法】用榛子基因组与转录组数据鉴定基因组中所有的SEP基因家族成员,并进行重要SEP基因的原核表达载体构建、基因表达及多克隆抗体制备,最后对参与胚珠发育调控的重要SEP蛋白进行免疫组织化学检测。【结果】(1)榛子基因组中鉴定到3条ChSEP蛋白序列(Cor0054610.1、Cor0008190.1、Cor0119400.1),所有ChSEP蛋白均包含SRF-TF与K-box结构域。制备了Cor0054610.1的多克隆抗血清E18305和E18306,它们的抗体抗原条带分子量在45 kDa左右,1∶1000稀释后可检测到1~2 ng抗原。(2)免疫组织化学分析结果表明:ChSEP在发育胚珠与败育胚珠的珠被中均有表达,珠被中的ChSEP表达量高于子叶,正常发育胚珠与败育胚珠ChSEP分布特征差异不明显。【结论】本研究在转录与蛋白水平上提供了ChSEP(Cor0054610.1)参与榛子胚珠发育调控的证据,为深入解析榛子胚珠发育机理提供了科学依据。[Objective]Normal ovule(kernel)filling is the basis of hazel kernel formation.Insufficient ovule filling can frequently lead to the shriveled kernels or empty nuts in hazel.SEP belonging to the E class MADS-box gene plays an important role in ovule development of plants.To investigate the important role of ChSEP in hazelnut ovule development,the key ChSEP genes involved in the development of hazelnut ovules were identified.Then,polyclonal antibody against ChSEP was prepared,followed by immunohistochemical analysis of ChSEP using developing and aborted ovules as study materials.The present study is beneficial for better understanding of ovule development mechanism regulated by SEP in hazelnut.[Method]All SEP gene family members in hazelnut genome were identified using genome and transcriptome data,and prokaryotic expression vector construction containing important SEP gene,gene expression,and polyclonal antibody preparation were carried out.Finally,important SEP involved in ovule development regulation was detected by immunohistochemistry.[Result](1)Three ChSEP sequences were identified in the genome of hazelnut,including Cor0054610.1,Cor0008190.1 and Cor0119400.1.All SEP proteins contained SRF-TF and K-box motifs.Polyclonal antisera of Cor0054610.1 was prepared,and the molecular mass of polyclonal antibody E18305 and E18306 was about 45 kDa,and 1−2 ng antigen could be detected after 1∶1000 dilution.(2)Immunohistochemical analysis showed that ChSEP was expressed in the integument of both developing and abortive ovules.The expression of ChSEP in integuments was higher than that in cotyledons.There was no significant difference in the distribution of ChSEP between developing and abortive ovules.[Conclusion]This study provides evidence that ChSEP(Cor0054610.1)is involved in the regulation of hazelnut ovule development at both transcriptional and protein levels,and provides a scientific basis for better understanding of the ovule development mechanism in hazelnut.

关 键 词：榛子 SEP 原核表达 多克隆抗体 免疫组织化学 

分 类 号：S722.37[农业科学—林木遗传育种] Q943.1[农业科学—林学]