基于转基因741杨与新疆杨杂交创制抗虫非整倍体毛白杨新种质OA北大核心CSTPCD
Creating a new germplasm of aneuploid Populus tomentosa with insect-resistance based on hybridization of transgenic 741 poplar and P.alba var.pyramidalis
[目的]以转BtCry3A基因三倍体 741杨为母本与二倍体新疆杨为父本进行人工杂交,以期快速获得非整倍体毛白杨抗虫优良新种质.[方法]采集转基因 741杨雌花枝、新疆杨雄花枝进行人工授粉杂交,收集即将脱落的花序,通过胚挽救技术获得杂交子代.通过聚合酶链式反应(PCR)检测杂交子代是否含有 BtCry3A基因;通过实时荧光定量PCR(RT-qPCR)对BtCry3A基因在母本与子代中的表达量进行分析.以二倍体新疆杨为参照,通过流式细胞技术检测杂交子代的倍性;并对子代幼苗表型进行初步观测分析.[结果](1)通过对未发育成熟种子进行胚挽救获得 8 个杂交子代,其中 6个杂交子代后续生长状态良好,5个子代遗传了母本的BtCry3A基因.(2)RT-qPCR检测显示,BtCry3A基因在5个子代中的表达量均高于母本,其中 3#表达量为母本 12 倍;(3)初步判定子代 1#为超四倍体,子代 2#、4#、8#为非整倍体,子代 3#可能为非整倍体或四倍体;(4)杂交子代表型差异大,5 个杂交子代叶形、叶片大小、节间距等均不相同,其中 3#和 8#生长势优于亲本.[结论]通过转 BtCry3A基因三倍体 741 杨与二倍体新疆杨杂交,快速获得了具有BtCry3A抗虫基因且发生形态变异的非整倍体毛白杨新种质,其中子代 3#因抗虫基因表达量显著高于母本,且生长势优于双亲,可作为优良潜在非整倍体毛白杨新种质进行后续抗虫性测试.
[Objective]Artificial hybridization was carried out using transgenic triploid 741 poplar with BtCry3A as the female parent and diploid Populus alba var.pyramidalis as the male parent,in order to quickly obtain new germplasm of aneuploid Populus tomentosa with insect-resistance.[Method]Collecting female flower branches of transgenic 741 poplar and male flower branches of P.alba var.pyramidalis,hybrid progenies were obtained by embryo rescue technique from the inflorescence that was about to fall off.The seedlings with BtCry3A were identified by polymerase chain reaction,and further expression of BtCry3A was measured via real-time fluorescent quantitative polymerase chain reaction.Subsequently,conducted ploidy analysis of these hybrid progenies using flow cytometry method.Finally,preliminary phenotypic analysis of the offsprings was performed.[Result](1)Eight hybrid individuals were successfully generated by combing artificial hybridization and embryo rescue techniques,among which six hybrid offsprings showed good growth status.Five offsprings inherited the BtCry3A gene from the mother plants.(2)The BtCry3A gene exhibited enhanced expression levels in the offsprings compared with maternal parent,with individual 3#demonstrating a 12-fold increase.(3)1#was hyper-tetraploid,2#,4#and 8#were aneuploid,while 3#may be aneuploid or tetraploid.(4)The hybrid offsprings exhibited diverse phenotype,the five hybrid individuals were different in leaf shape,leaf size and pitch spacing,with 3#and 8#showing superior growth vigor compared with parental lines.[Conclusion]Employing artificial hybridization between triploid 741 poplar with BtCry3A and diploid P.alba var.pyramidalis,we creat the novel germplasms of aneuploid P.tomentosa with BtCry3A and diverse phenotypic variation.Offspring 3#has significantly higher anti-insect gene expression than their mothers,and growth potential is better than parents.It can be used as an excellent potential aneuploid hair poplar new germplasm for subsequent insect resistance tests.
齐婉芯;陈婷婷;宋佳力;安新民
北京林业大学生物科学与技术学院,林木遗传育种全国重点实验室,林木育种与生态修复国家工程中心,林木花卉遗传育种教育部重点实验室,树木花卉育种生物工程国家林业和草原局重点实验室,北京 100083
林学
毛白杨抗虫人工杂交非整倍体新种质创制
Populus tomentosainsect-resistanceartificial hybridizationaneuploidnew germplasm creation
《北京林业大学学报》 2024 (012)
92-102 / 11
科技创新2030—重大项目课题(2022ZD0401503).
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