Genetic Analysis And Clone Of Pbh Gene For Purple-Black Hull In Rice (Oryza Sativa L.)

The color of rice hull is one kind of visual traits which can be easily identified after heading. Although the hull color genes have been widely studied, but there are no research about the pigment in rice hull related flavonoid biosynthesis pathway. In this study, a natural rice mutant pbh with purple-black hull was isolated by investigation the pigment of rice hull at mature time. Moreover, the gene responsible for the phenotype was fine mapping. We also investigated the genes expression pattern invloved in flavonoid pathway. The main conclusions are as follows:1. The pbh mutant came from the high generation of hybrids groups between 05261 and 9311, is a natural spontaneous mutation. All seed hulls color of the pbh panicles kept green before the dough-ripe stage (20 days after flowering), till the ripe stage all of the full seeds become purple-black and at the same time the purple-black grains with seed-shattering habit. The maturing spikelets present the top seeds hull were purple black and the bottom were green. We used the purple-black mutant cross with the normal variety hull color Balilla (pbh×Ballila). By investigation, the pbh mutant was controlled byo single dominant gene in this genetic background. We found that there was another gene controlled the seed pericarp color in our materials, but it did not separate together with the phenotype of coloration in hull. Thus, we thought the genes associated with the color of hull and pericarp in the seed can be seen as two independent genes for studying. Because all of the segregated purple-black hulls grains are easily shattering, we hypothesized that seed-shattering habit of grains may be closely linked to purple-black hull or determined by one same gene for variety of traits. Comparison of agronomic traits between homozygous pbh and WT, despite of slightly longer panicle and 1000-grain weight increment, no significant difference on other traits was observed.2. By analysis anthocyanins and PAs content and composition in the rice hull at maturity with UV spectrophotometer and HPLC, we found that soluble proanthocyanin significantly decreased in pbh mutant, but no remarkable differences in anthocyanins. HPLC analysis of PAs extracts revealed all the peaks were higher in wild than mutant. The abundance of unpolymerized epicatechin was also observed lower in mutant than that in wild type.3. The Pbh gene was finally delimited to a 64.9 kb region, and there were 12 predicted genes in this region. By sequencing these genes, we found ORF1 encoded MRP (multidrug resistant protein) exhibited a single sequence difference (C→T) in 10 exon between the wild type and pbh mutant, which changes Valine to Isoleucine, we named this gene as OsMRP. Besides, a 22 bp insertion in pbh mutant was also found in the gene ORF11, which encoding an amino acid transporter protein (designated as Bh4-5), and this gene had been suggested as Bh4 allelic gene.4. For confirmation the gene which controlled purple-black hull, we have constructed complementary, over-expressed and RNAi vectors about OsMRP and Bh4-5 for futher studying. Unfortunately, it was difficult to go on genetic transformation because of the callus tissues in pbh and wild-type were hardly to culture, so we had not got the over-expressed and complementary transgenetic plants untill now, which using pbh mutant and wild-type as receptor materials. Only got some transgentic plant of RNAi vector which using Lijiangxintuanheigu, Nipponbare, kitaaki as receptor material.5. By analysis the expression of related genes in flavonoid pathway, we found that the key structural genes OsCHI, OsDFR, OsF3H, OsF3'H, OsGT were up-regulated in pbh mutant different tissues at maturity. OsGST, OsAHA10, OsMATE involved in the flavonoid transport pathway mainly expressed in pbh mutant, but the same gene in plants local tissue (leaves or stems) turned around a high expression of wild-type. OsFLS, OsLAC and OsLAR at the seedling stage of the wild type and mutant had no significant difference in expression, but in the mature mutant leaves OsFLS, OsLAR higher than wild-type, while the stem was lower. OsPAL had a high expression in various tissues of wild-type and pbh mutant, but no significant difference. Surprisingly, OsANR which control the PAs precursor biosynthesis was significantly higher expressed in wild-type than mutant in every tissuse. These results suggested that Pbh gene might affected the genes expression which invloved in PAs pathway, further illustrated the pbh mutant is a flavonoid biosynthetic mutant.6. From the results of the genes expression analysis at mature hulls by qPCR, we found that most of the selected genes including OsDFR, OsCHS, OsF3H, OsF3'H, OsAHA10, OsANS, and OsGT were up-regulated expression in the hull of pbh, while the expression amounts of genes OsPAL, OsMATE, OsCHI, OsLAR and OsFLS revealed no statistical differences. However, two PAs synthesis pathway genes, OsLAC and OsANR, exhibited significantly down-expression in pbh mutant.We hypothesized that mutations in the gene may be a positive regulator of flavonoid pathway genes, while the negative regulation of two genes in proanthocyanidin biosynthesis pathway.