| Rapeseed is one of the most important oil crops in China. Rapeseed heterosis utilization is the most efficient way to increase yield, improve resistance and tolerance. Recessive genic male sterility (RGMS) is an efficient way to utilize heterosis in Brassica napus. The male sterility RGMS two-type line S45AB is controlled by the two duplicate recessive genes (Bnmsl, and Bnms2), when we use it to product hybrid seed, the50%fertile plants in mother line are required removal, which limits its wide application. So the major problem of using heterosis of this RGMS is how to remove the50%fertile plants from the female line. So, the major goal for this research is to create a100%male sterile population based on the GMS material. The main results are as follows:1. Cloning the AtLEC1and constructing an inducible marker-free vector and its transformationAccording to the genome sequences of Arabidopsis thaliana, the proper primers were designed to amplify the AtLEC1gene from Arabidopsis. Compared with the published genome sequences (TAIR, The Arabidopsis Information Resource), the cloned AtLEC1sequences had the base pair A/T at the669th site instead of the G/C, but the amino acid sequence was the same, so this cloned AtLEC1gene can be used for the following experiments.According to the improved methods of building ecdysone induction expression system (Venkata et al.,2006), the AtLEC1, the main induced components of the system and the restore gene BnCYP704B1totally the three elements were simultaneously inserted into the original pCAMBIA2300and the modified vector pCAMBIA2300was used to remove the marker gene. The two vectors were transformed to A.thaliana and B. napus individually. In the transformation of A. thaliana, there were17seedlings shown the resistance, and9of them were positive in To generation. The transformation rate is about54.1%. By using specific primers, we obtained6transgenic plants which contained the marker genes. 2. Induced expression of the AtLEC1gene in A.thaliana and B. nap usWe got the T1seedlings by harvesting the9positive Arabidopsis thaliana To plants. Each line was sprayed by four concentrations of inducer methoxy insects hydrazide (OnM,1000nM,5000nM,10000nM), after that we continuously observed the plant growth status, the majority of individual grew well with no necrotic spots. However, there was a significant difference between one sprayed line and wild-type under three concentrations gradient (1000nM,5000nM and10000nm).In induced expression experiments with transgenic Brassica napus, two methods were adopted as follows:(1) filter paper germination test, we soaked the filter paper in an inducer methoxy insects hydrazide with four concentrations (same as above), sow To seeds on the filter paper. All seeds of the6lines can germinate and grow in the presence of methoxyfenozid, there was no necrotic spots on the leaves.(2) field inducing-test, we sprayed the plants of the6lines with four concentrations of methoxyfenozide solution (same as above) once per three days for20days. All seedlings can grow normally in the presence of methoxyfenozide, and no necrotic spots on the leaves and the plants have normally flower and silique.When spraying inducer onto the seedlings of Brassica napus, we sample3lines of B. napus randomly for testing the amount of target gene expression which was under the same concentration but different time interval and the same time interval but different concentration. The results show that the AtLECl gene activity began to increase at6and8days after spraying, and also they showed little induced the AtLECl gene activity at10000nM concentration of methoxyfenozide after20days. |
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