摘要
水稻(Oryza sativa L.)是世界上最重要的粮食作物之一,全世界约有二分之一以上的人口以稻米为主食。直链淀粉含量(AC)的高低是影响稻米食用品质的一个重要因素。利用基因工程技术对稻米淀粉合成相关基因的表达进行调控,可以改良稻米的淀粉品质,提高其食用品质或加工品质,创造出不同直链淀粉含量的新型种质资源。
本论文以籼型杂交稻亲本LP03和P88S为受体材料,研究对AC含量有重要影响的蜡质基因(Waxy)的过量表达与反义RNA干扰对其直链淀粉含量的效应,并对影响遗传转化效率的主要因素进行了探讨。主要结果如下：
1.建立了适合于供试材料的愈伤组织培养体系。LP03、P88S成熟胚愈伤组织的最适诱导培养基和继代培养基分别为N6D2.5(N6盐分和维生素+2.5mg·L-12,4-D+0.2mg·L-16-BA),NBD2.5(N6盐分、B5维生素+2.5mg·L-1 2-4,D+0.2mg·L-16-BA)。成熟胚愈伤组织继代培养7天后的转化效率高。
2.优化了适宜供试材料的农杆菌转化体系。农杆菌菌液浓度OD600为0.8,侵染时间15 min,共培养两天为本试验合适的转化参数。
3.获得了含有目的基因的水稻再生植株。采用农杆菌介导法将正义Waxy基因和反义Waxy基因分别导入供试材料中,获得了转基因再生植株,PCR检测表明外源基因已经整合进再生植株的基因组中,并初步分析了To代种子中的AC含量。
4.系统分析了愈伤组织中矿质元素的动态变化特性。对成熟胚愈伤组织诱导和继代过程中种胚、各阶段愈伤组织、愈伤组织形成的芽中矿质元素的变化特性进行了分析,结果表明矿质元素含量对愈伤组织的诱导率及其胚性生长具有显著作用。芽中低浓度的元素含量及种胚中高浓度的元素含量对愈伤组织诱导的启动有决定性作用。较高的K、P、Fe、Ca含量有利于愈伤组织诱导,较高的Fe、Cu含量容易导致继代过程中愈伤组织的褐化。
Rice(Oryza sativa L.)is one of the most important food crops in the world,and it is also the staple food for more than fifty percent population all over the world. Amylose content in rice endosperm is one of the key factors that affects the rice eating and cooking quality. Regulating the expression of amylose systhesis interrelated genes by genetic engineering could improve the eating and cooking quality of rice and create new resources with different amylose content.
In this paper, sense Waxy gene and anti-sense Waxy gene were transformed respectively into the calli of hybrid rice parent lines LP03 and P88S for overexpression or RNA interference analysis, meanwhile the factors influencing the rate of transformation were studied. The results are as follows:
1、Callus culture system have been established. The results showed that N6D2.5(N6 salinity,vitamin+2.5mg-L-12,4-D+0.2mg-L-16-BA), NBD2.5(N6salinity, B5vitamin +2-4,D 2.5mg-L-1+0.2mg-L-16-BA)were the best suitable medium for LP03,P88S calli induction and subculture respectively. The transformation efficiency was higher after subcultured 7 days.
2、The transformation system suitable for these hybrid rice parent lines was optimised. It was found that the optimal conditions for higher transformation efficiency were as follows:OD6oo value 0.8,the treatment time 15min and co-cultivation for 2d under dark after preculture.
3、Transgenic plants with Waxy gene have been obtained. Many transgenic rice lines transformed with the sense Waxy gene and anti-sense Waxy gene respectively were regenerated, and the integration of these transgenes in the transformed plants were confirmed by PCR. The amylose content in the endosperm of mature seeds from To transgenic plants with Waxy gene was analyzed.
4、The dynamic character of mineral element content in different parts were analyzed during the induction and subculture. Results showed that the content of mineral elements had significant effects on the rate of callus induction and embryo development. Lower concentration of mineral elements in buds and higher concentration of mineral elements in embryos was c(?)ucial for callus induction. The higher content of P,K,Fe,Ca were beneficial to the induction of callus. The higher content of Fe,Cu was responsible for browning phenomenon in subculture.
引文
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