北美鹅掌楸LtMYB305基因的克隆及功能分析  

Cloning and Functional Analysis of LtMYB305 in Liriodendron tulipifera

作　　者：刘换换 杨立春 李火根 LIU Huan-huan;YANG Li-chun;LI Huo-gen(College of Forestry,Nanjing Forestry University,Nanjing 210037;School of Landscape Architecture,Jiangsu Vocational College of Agriculture and Forestry,Zhenjiang 212400)

机构地区：[1]南京林业大学林学院,南京210037 [2]江苏农林职业技术学院风景园林学院,镇江212400

出　　处：《生物技术通报》2024年第4期179-188,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32201591,31470660)。

摘　　要：【目的】MYB305作为MYB家族R2R3亚族成员,对植物花蜜腺发育、花蜜蛋白表达、淀粉积累和水解、类黄酮合成发挥重要作用。因此,研究MYB305的表达模式及功能对于北美鹅掌楸花蜜腺调控分子机理研究具有重要意义。【方法】本文以蜜源植物北美鹅掌楸的花蜜腺为材料,通过分光光度计法测定5个时期花蜜腺的花青素含量,采用RACE技术克隆LtMYB305基因,利用RT-qPCR技术检测了该基因在北美鹅掌楸不同组织间相对表达量,以烟草叶片为材料验证LtMYB305蛋白定位,并以野生型拟南芥为材料进行遗传转化实验,研究LtMYB305基因功能。【结果】北美鹅掌楸花蜜腺的花青素从膨大后期开始积累,初放期含量急剧增加,败花期含量最高,达27.14μg/g,与花蜜分泌、着色过程相一致。LtMYB305基因全长为931 bp,编码了198个氨基酸,其蛋白为亲水性蛋白和非跨膜蛋白;LtMYB305仅在北美鹅掌楸盛花期花蜜腺中高水平表达,在其他组织中几乎不表达。LtMYB305蛋白定位于细胞核。过表达LtMYB305基因的拟南芥出现侧蜜腺的“蜜腺沟”消失和加深表型,与花蜜腺相关基因AtMYB305、AtPIN6和AtSWEET9表达量上调,AtCRC、AtBOP1/2、AtMYB21、AtSWEET3/4/7基因表达量下调。【结论】LtMYB305具有典型转录因子特征,并且参与调控拟南芥花蜜腺的发育。【Objective】MYB305 as a member of the MYB R2R3 subfamily,plays an important role in plant nectary development,nectar protein expression,starch accumulation and hydrolysis,and flavonoid synthesis.Therefore,it is of great significance to study the expression profile and function of MYB305 for the molecular mechanism study of Liriodendron tulipifera floral nectary.【Method】Having floral nectary of nectariferous plant L.tulipifera as materials,the anthocyanidin content in five stages of floral nectary was detected by spectrophotometry,LtMYB305 gene was cloned via RACE(rapid-amplification of cDNA ends)technology,and relative expressions among different tissues were analyzed by RT-qPCR assay.Then,subcellular localization of LtMYB305 protein was verified using tobacco leaves as materials,and the function of LtMYB305 gene was studied by genetic transformation approaches of wild type Arabidopsis thaliana(col).【Result】The anthocyanidin in the nectaries of L.tulipifera was accumulated from the late expanding stage,increased sharply at the early flowering stage,and reached the highest of 27.14μg/g at the withering stage,which was consistent with the process of nectar secretion and coloring.The full-length of LtMYB305 was 931 bp encoding 198 amino acids and its protein was hydrophilic and non-transmembrane.LtMYB305 was expressed highly only in the nectary of L.tulipifera at anthesis stage,and hardly in other tissues.LtMYB305 protein was localized in the nucleus.After the overexpression of LtMYB305,the nectary groove of the lateral nectary disappeared and deepened,the expressions of AtMYB30,AtPIN,AtSWEET9 genes related to flower nectary were up-regulated,and the expressions of AtCRC,AtBOP1/2,AtMYB21,AtSWEET3/4/7 genes were down-regulated.【Conclusion】The LtMYB305 has typical characteristics of transcription factor and is involved to the nectary development in L.tulipifera.

关 键 词：北美鹅掌楸 蜜腺 MYB305 基因克隆 功能分析 

分 类 号：S792.21[农业科学—林木遗传育种] Q943.2[农业科学—林学]