| Rice is one of the most important food crop, and its output level is directly related to food security, so in-depth study of rice yield has important implications. At present, the controlling mechanism of rice yield traits has made great progress, particularly in grain weight and effective panicles. In contrast, little is known about the rice setting rate, another important factor of rice yield. For the improvement of setting rate effectively increase grain number and yield, further study of rice seed setting rate has important significance both for its mechanism and for the improvement of rice yield.We have recently cloned a major gene, POLLEN TUBE BLOCKED 1 (PTBl), which controls panicle seed setting rate (PSSR), by using a low PSSR mutants Map-based cloning found that a 470bp deletion in the coding region of PTB1, it encodes a RING-type E3 ubiquitin ligase, which promotes pollen tube growth by controlling the amount and depth of growth of pollen tubes in the rice reproductive tract, which directly regulate the level of rice PSSR. We also found that rice PSSR decline under abnormal conditions, such as high or low temperature, mainly due to the down regulation of expression level of PTB1 under certain abnormal temperature conditions. Further investigations showed PTB1 protein has E3 ubiquitin ligase biochemical function in vitro, Thus, we propose that PTB1 recruits its substrate (an unknown inhibitor of PT growth) for target degradation, thereby promoting PT growth by controlling the amount and depth of growth of PTs and, subsequently, positively affecting rice PSSR and ultimately improving grain yields.Several genes’cloning suggest that most rice yield-related genes are plentyful in allic variations, and to some extent, refrect the domesticated characteristics of cultivated rice. PTB1 is the first reported gene on rice PSSR controling, however, its natural variation characteristics and whether it is related to the domestication is still unknown. Therefore, in the present study, we clone the PTB1 gene and its regulatory sequences from a collection of word-wide rice germplasm(including wild rice, japonica subspecies and indica subspecies), with the aim of revealing the allelic variation of PTB1 gene and its association with the PTB1 expression and rice PSSR. Also, we want know whether the PTB1 gene is related to the rice domestication process. The main results are as follows:1. Cloning and sequencing of PTB1 gene from a total of 107 rice material showed that PTB1 has abundant natural variations, among which 32 SNPs loci and 5 InDel sites were found:19 SNPs mutation sites and 4 InDel sites in PTBl promoter and 13 SNPs loci and 1 InDel site in its exons.2. PTB1 gene sequencing results of all rice materials were statistically analyzed and totally 16 haplotypes were found. Sequence variation analysis completely identified 16 haplotypes in O. rufipogon (21), however, only 3 haplotypes were present in O. sativa(86). The PTB1 haplotype significantly reduced from O. rufipogon to O. sativa, suggesting that the PTB1 locus was selected by ancient humans during rice domestication, means that PTB1 is a domesticated related gene. The genetic parameter calculation results also confirmed this conclusion.3. A population investigation revealed a clear bias for both sub-species of Oryza sativa:approximately 98% of indica rice cultivars were in haplotypes of Type 12 or 16 (two highly similar types with few SNPs within the promoter), whereas approximately 75% of japonica rice cultivars were in the substantially distinct Type 1, suggesting that PTB1 was an indica-japonica-differentiated gene.4. Previous studies have showed that PTBl gene expression was associated with the PSSR of transgenic plants. We then carried out PSSR and PTB1 gene expression investigations among several rice natural materials. We found that PTB1 relative expression level in different materials is closely related to the rice seed setting rate(r=0.74, P<0.01). The results is similar to the previous conculsion within the transgenic plants group. Our results suggest that the natural variation of rice seed setting rate is directly related to natural variation of PTB1 expression level.5. Using the natural allelic variation obtained and the corresponding expression data, we further studied whether the PTB1 allelic variation is directly related to the gene expression. Correlation analysis results showed that,2 haplotypes(CACC and TCGT),which were determined by 4 SNPs(-1156,-1069,-931 and -621) located in the promoter were significantly related to PTB1 expression(P<0.05,t-test) and to rice PSSRs(P<0.05, t-test). The results showed that PTB1 natural variation located in the promoter determines the PTB1 expression, which directly determines the PSSR, which suggest the natural variation in promoter region is the main reason of PSSR variation of rice materials.In summary, the present study shows that PTB1 has abundant natural variations, and these variations are related to the indzca-japonica-differentiatiation and the cultivated rice domestication progress. Meanwhile, the PTB1 natural variation located in the promoter determines the PTB1 expression, which directly determines PSSR, which suggest the natural variation in promoter region is the main reason of PSSR variation of rice materals... |
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