摘要
愈伤组织瞬时转化体系可初步快速地验证基因功能和鉴定相关表型。为提高板栗愈伤组织瞬时转化体系的转化效率和稳定性,并鉴定板栗淀粉合成酶基因的功能,该研究以板栗‘燕山红栗’幼胚胚芽诱导形成的愈伤组织为材料,通过不同状态的愈伤组织和表达载体优化板栗愈伤组织瞬时转化体系;并构建板栗淀粉合成关键酶基因CmSSⅠ沉默载体,验证CmSSⅠ基因在板栗中的功能。结果表明:(1)愈伤组织可按照体细胞胚起动难易程度划分为胚性愈伤组织(EC_E)和胚性愈伤组织早期阶段(EC_DⅠ、Ⅱ、Ⅲ),且白色、外表相对分散的胚性早期阶段愈伤(EC_DⅢ)更适合板栗瞬时转化体系。(2)转化材料为胚性愈伤早期阶段(EC_DⅢ)时,RNAi沉默载体pK7GWIWG2(Ⅱ)RR-277的瞬时转化效率高达87.67%。(3)通过最适愈伤组织瞬时转化体系,基因CmSSⅠ在转基因愈伤组织中表达显著下调,且沉默CmSSⅠ阳性愈伤组织的总淀粉和支链淀粉含量显著降低,直链淀粉变化差异不显著。该研究提高了板栗愈伤组织瞬时转化体系的转化效率和稳定性,证明基因CmSSⅠ正调控板栗淀粉的合成,这为板栗基因功能的研究奠定了基础,进一步为分子辅助育种提供了技术平台。
The transient transformation system plays an important role for initially and rapidly verifying gene functions and identifying related phenotypes.To improve the transformation efficiency and stability of transient transformation system of the Chinese chestnut callus and to identify the function of the Chinese chestnut starch synthase gene(CmSSⅠ),we optimized the transient transformation system of Chinese chestnut callus through different states of callus tissues and expression vectors by using the callus tissues induced from young embryos of Chinese chestnut‘Yanshanhongli’.The results showed that:(1)callus was divided into an embryogenic callus(EC_E)and early stage of embryogenic callus(EC_DⅠ,ⅡandⅢ)based on the difficulty level of initiating somatic embryo.The early stage of embryogenic callus with white and relatively dispersed texture(EC_DⅢ)were more suitable for the transient transformation system of Chinese chestnut callus.(2)The transient transformation efficiency of RNAi silencing vector pK7GWIWG2(Ⅱ)RR-277 was the highest at 87.67%when the transformed material was at the early stage of embryonic callus(EC_DⅢ).(3)The expression of CmSSⅠgene was significantly down-regulated in the transgenic callus tissues by the optimal callus transient transformation system,and the contents of total starch and amylopectin of the CmSSⅠ-silenced positive callus were significantly reduced,and amylose content was not significant differences.In this study,the efficiency and stability of the transient transformation system of Chinese chestnut callus tissues were improved,and it was demonstrated that the gene CmSSⅠwas regulating the synthesis of Chinese chestnut starch.This laid a foundation for the study of Chinese chestnut gene function and further provided a technical platform for molecular assisted breeding.
作者
于文杰
楚天舒
宋丽
葛婧怡
秦岭
邢宇
YU Wenjie;CHU Tianshu;SONG Li;GE Jingyi;QIN Ling;XING Yu(College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2022年第1期154-161,共8页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(3187031020)。
关键词
板栗
愈伤组织
瞬时表达
遗传转化
CmSSⅠ
Chinese chestnut
callus
transient expression
genetic transformation
CmSSⅠ
