转反义蜡质基因纯合株系间直链淀粉含量与RVA谱关系分析

 【目的】排除以往水稻亚种或品种间RVA谱分析时存在着的遗传背景差异,分析转反义蜡质基因不同直链淀粉含量纯合株系间RVA各特征值的差异,更清楚地揭示直链淀粉含量与RVA谱特征值的关系。【方法】采用根癌农杆菌介导共转化和花药培养相结合的方法,培育含有反义蜡质基因的花培纯系,并对各株系进行直链淀粉含量和RVA谱测定和分析,通过比较RVA谱曲线图形及RVA谱特征值的显著性测定,分析相同遗传背景条件下直链淀粉含量对各RVA谱特征值的作用。【结果】在武运粳7号转基因后代中,获得了只含反义蜡质基因、不含潮霉素抗性标记的花培株系77个,经T1H1和T1H2二代直链淀粉含量测定,有19个株系的直链淀粉含量接近对照（对照16.0%）;有50个株系直链淀粉含量比对照下降1%～5%,其中,直链淀粉含量在11.0%～13.9%有30个株系;另外,有8个株系的籽粒为蜡质状,直链淀粉含量降至3.1%～4.0%。不同直链淀粉含量纯系间RVA谱分析发现存在二种RVA谱曲线图形,直链淀粉含量在3.1%～4.0%株系明显不同其它株系,也不同于常规糯稻;对RVA谱曲线图形相同、直链淀粉含量不同的株系进行分类比较,低直链淀粉含量株系的RVA谱曲线最终表现为曲线逐步上升,但没有超越第1个峰值。对不同直链淀粉含量株系类群的各RVA谱特征值进行显著性测定,3.1%～4.0%株系群有5个特征值与其它二个株系群存在极显著差异;11.0%～13.5%株系群有3个特征值与对照16.0%组存在极显著差异;直链淀粉含量与RVA谱各特征值之间优化数学模型显示：峰值时间和消减值与直链淀粉含量关系较为密切。【结论】导入反义蜡质基因,能导致直链淀粉含量降低;通过花药培养能快速获得纯合株系;直链淀粉含量差异不仅影响到各RVA谱特征值,而且还造成RVA谱曲线不同。

Analysis of Relationship Between RVA Profile and Amylose Content of the Transgenic Rice with Antisense Waxy Gene

【Objective】 Previous studies focused on the analysis of RVA profile in rice at the level of the subspecies or varieties (Oryza sativa L.) in which there were differences in genetic background. In this study, antisense waxy homozygous transgenic lines were used to analyze the differences in RVA characteristic values among the lines with different amylase contents in an effort to eliminate the influence of different genetic backgrounds and to more clearly understand the relationship between the amylose content and RVA characteristic value. 【Method】 Anther culture pure lines containing antisense waxy gene were generated by Agrobacterium tumefaciens mediated co-transformation and followed by anther culture in transgenic rice variety. The amylose content and RVA profile were determined in the anther culture lines, and the effects of the amylose content on RVA characteristic value were analyzed under the same genetic background conditions through the RVA profile graphic comparison and the significant determination of RVA eigen value. 【Result】 Seventy-seven pure lines of anther culture that contained only antisense waxy gene but no hygromycin resistance marker were obtained from the transgenic plant offspring of Wu Yunjing No.7 rice variety (O. sativa L. subsp. japonica cv.). Through the dertermination of the amylose content to the T1H1 and T1H2, it was showed that 19 lines had the levels of amylose content similar to the control (16.0%), and that 50 lines were 1%–5% lower than that of control in the amylose content, and some of 30 lines had the amylose content within 11.0%﹣13.9%, and that 8 lines had the amount of amylase content reduced to 3.1%–4.0%. The results of the analysis of the rice starch RVA profile in the pure lines with different amylose contents indicated that there were two kinds of RVA profile: the lines with 3.1%﹣4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The results of comparison of the lines with same RVA profile to the lines with different amylose content, also showed that the lines with lower amylose content had a distinguished RVA profile with the curve of increasing gradually in finally, but no exceeding the first apex. In significance tests for the characteristic values of RVA profile in the lines with different amylose content, there were five characteristic values of RVA remarkably different among the lines with 3.1%–4.0% of amylose content compared with two other groups, and that there were three characteristic values remarkably different among the lines wiht 11.0%﹣13.5% of amylose content compared with control group. An optimized mathematical model, which characterized the relationships between the amylose content and RVA eigenvalue, was established in this study. From this model, it clearly indicated that the parameters PeT and SBV were more related to the amylose content. 【Conclusion】 Introduction of antisense waxy gene could lead to a reduced amylose content in rice. Anther culture could result in producing pure lines more quickly. The amylose content differences not only affected the characteristic value of RVA but caused different RVA profile as well.