Study On Identification And Regulation Mechanism Of Related Genes For Pod Shatter Resistance In Brassica Napus L.

In oilseed, pod shattering can easily cause yield loss, especially with the use of combine machine at harvest. It is very important to establish a reliable and handy method to assess pod shatter resistance for the development of pod shatter resistant lines. The advance on molecular biology opens up the possibility for the development of oilseed rape lines resistant to pod shattering by genetic engineering. Understanding of the molecular mechanism underlying pod shattering can facilitate the improvement of pod shatter resistance of Brassica napus varieties.1. Assessment method for pod shatter resistanceIn order to obtain a reliable assessment method, different methods for determining pod shatter resistance were compared and the effects of the moisture content of pods, impact conditions on determination accuracy were analyzed. The results showed that random impact test method was more suitable for the determination of pod shatter resistance. Measures such as using pods stored in an airproof bag overnight after baked at80℃for30min to equilibrate moisture, record cracked pods after every two min shaking, as well as reduce the number of impact balls to8could increase the accuracy and efficiency, resulting in better pod shatter resistance index (SRI) distribution of involved varieties. One hundred and three rapeseed newly bred lines which were involved in the national variety trials in2010were assessed by the improved random impact test method, the results showed that the average value of pod shatter resistance index was0.148, with a variation of0-0.79. The modification of random impact test method had advantages over the original one in terms of repeatability, accuracy and efficiency, which was suitable for selection of pod resistant lines in rapeseed breeding programs and quantification of pod shatter resistance of lines involved in genetic as well as molecular analysis.2. FRUITFULL gene functional analysisFRUITFULL (FUL) gene was analyzed to understand the molecular bias of pod shatter resistance in alloploid B. napus varieties. Both the resistant and susceptible varieties possessed the same two FUL gene loci on A and C genomes, BnaA.FUL.a and BnaC.FUL.a, respectively. In resistant variety R1-1, only BnaA.FUL.a expressed, BnaC.FUL.a was silenced. Ectopic over-expression of BnaA.FUL.a in A. thaliana resulted in elevated resistance to pod shattering, as well as earlier flowering and three pods on a single peduncle. In the BnaC.FUL.a ectopic over-expressed A. thaliana plants, the pods failed to shatter too, but no early flowering and other phenotype variation were observed. These results demonstrated that the genomic divergence and expression differentiation of BnaX.FUL gene had great significance for polyploidy evolution and phenotypic variation. BnaA.FUL.a had a more distinct effect for pod shatter resistance.3. PECTATELYASE gene functional analysisA PECTATELYASE gene (BnPL) was cloned in Brassica napus.The result demonstrated that BnPL played an important role in plant germ development, especially for the development of flower and pod. In over-expression BnPL transgenic plants, there were three different variety phenotypes. The first result was about the flower for petal defect and sterility. The second was that the width of the fruit in transgenic plants was larger than in the wild plants. The last was that there was a deep gap in DZ in transgenic plants. In anti-expression BnPL transgenic plants, the size of the fruit in transgenic A. thaliana was smaller than in the wild plants and no pod shattering for the pods in anti-expression BnPL transgenic plants. These results showed that BnPL could effect the cell separation. It existed cell separation and degradation when pod shattering happened in oilseed, so pod shatter resistance could be improved by anti-expression BnPL with genetic operation in oilseed plants.4. Identification of differentially expressed mRNAs and miRNAsTo identify mRNAs and miRNAs for pod shatter resistance in Brassica napus, mRNAs and miRNAs expression levels of fruits in two oilseed rape varieties with distinct levels of pod shatter resistance were performed with Illumina sequencing system. The results revealed608mRNAs and57miRNAs which were differentially expressed in the siliques of R1-1and R2. From the608differentially expressed mRNAs, some of them belonged to the endo-polygalacturonase (PG), pectin lyase-like gene superfamily, pectinacetylesterase gene family, peroxidase activity, phytohormone genes, and so on. The inversely correlated expression miR156and its target gene SPL (Bra004674) in R2and R1-1implied their possible regulatory role for pod shattering in B, napus. These results provided some novel information on molecular mechanisms of pod shattering and a useful reference for further research on the improvement of pod shatter resistance in B. napus.