籼稻转反义蜡质基因后代的直链淀粉含量测定和纯系选育

DETERMINATION OF AMYLOSE CONTENT IN TRANSGENIC PROGENIES CONTAINING AN ANTISENSE WAXY GENE AND IDENTIFYING OF PURE LINES IN INDICA RICE

通过根癌农杆菌介导将反义蜡质基因导入籼型雄性不育保持系龙特甫B中,获得30个PCR检测为阳性的转基因植株,其中,28个为Southern检测阳性。T_1种子直链淀粉含量测定结果表明,有21个转基因植株比龙特甫B明显下降,下降幅度为3%-13%,并在部分转基因植株的种子中观察到蜡质状籽粒;对6个转基因植株进行了不同世代的直链淀粉含量测定,在L3和L5的T_4代中,选择到直链淀粉含量分别为15.9%和8.4%的纯合株系,经凝胶电泳测定,W_x蛋白量明显降低,并与直链淀粉含量下降表现一致。以L3-1-1-1(15.9%)和L5-8-2-1(8.4%)纯合株系为亲本,分别与龙特甫A进行成对杂交和回交,并测定了F_1和B_1F_1种子直链淀粉含量,以L3-1-1-1作亲本的F_1为21.4%,B_1F_1为17.1%;以L5-8-2-1作亲本的F_1为13.6%,B-1F-1为9.3%,结果表明:在不育系的转育过程中,以中低直链淀粉含量的转基因纯合株系为亲本,能有效降低F_1和B_1F_1的种子直链淀粉含量。

Through genetic transformation mediated by Agrobacterium tumefaciens,an anti sense waxy gene was introduced into Longtefu B line,a male sterile maintainer line in indica (Oryza sativa L.).Thirty transgenic plants showed integration of antisense waxy gene into the genome as determined by PCR assay,and twenty eight were confirmed by Southern blotting.T_1 seeds from twenty one transgenic plants showed a marked decrease of amylose content,ranging from 3%-13% less than control,and seeds from some transgenic lines exhibited typical waxy phenotype.Six transgenic lines were selected to examine the amylose content in different genera- tions.In the T4 generation,two homozygous lines,Long 3-1-1-1 and Long 5-8-2-1,were selected, with amylose content of 15.9% and 8.4% respectively.The amylose content in these lines is in consistent with the decrease of the accumulation of Wx protein as determined by SDS-PAGE elec- trophoresis. Cross and subsequent backcross of Long 3-1-1-1 and Long 5-8-2-1 with the Longtefu male sterile line were performed to determine the changes of amylose content in F_1 and B_1F_1 seeds. The results showed an average amlyose content of 21.4% in F_1 seeds with Long 3-1-1-1 as a parent,while only 13.6% with Long 5-8-2-1 as a parent.In addition,the average amylose con- tents in B_1F_1 seeds were 17.1% and 9.3% respectively.Our results indicated that during the fertility transfer in the male sterile line,stable transgenic lines with medium or low contents of amylose had direct effects on amylose content in F_1 seeds.