MAP-Based Cloning And Functional Analysis Of Two Genes Related To Chlorophyll-Deficiency In Rice

The chloroplast, the plant photosynthesis organelle, is essential for carbon assimilation and amino acid synthesis. Rice chlorophyll (Chl)-deficient mutants are suitable materials for the research in photosystem of plants, the development of chloroplast and chlorophyll biosynthesis, the structure and function of chloroplast. Additionally, rice Chl-deficient mutants can be used as marker in utilizing rice heterosis. Therefore, it is very important to identify Chl-deficient mutant and clone these genes controlling Chl-deficient trait.In the research, we analyzed two rice Chl-deficient mutants, green-revertible albino 1 (grcl) and green-revertible chlorina 1 (grc1). We studied the phenotype of the mutants, the electron microscopy of the chloroplast, the cloning and function of genes. The research provided the basis on understanding the molecular mechanisms of chlorophyll biosynthesis and chloroplast biosynthesis, and materials in utilizing rice heterosis.1. The main results of GRA1 gene are as follows:(1) Rice green-revertible albino 1 (gral) mutant was isolated from a Co-irradiated mutant pool of indica rice cultivar 9311. The mutant showed albino in the first and second leaf, and gradually turned into green about four weeks after seeding. Ultrastructure of chloroplasts analysis showed that the numbers of chloroplasts decreased, loosed thylakoid lamellar structures and more osmiphilic bodies in gral albino leaves. Additionally, agronomic traits such as plant height, number of tillers, panicle length, rate of setting and 1,000 seeds weight showed no obvious difference between gral and wild type. To identify whether the phenotype of mutant grcl was affected by temperatures, we measured the contents of chlorophyll a and chlorophyll b under six temperatures 20,24,28,30,32,36℃. Compared to wild type, the contents of chlorophyll a and b were significantly decreased under 20,24 and 28℃ temperatures. But the contents of chlorophyll a and b were no significant difference between mutant grcl and wild type under 30,32 and 36℃ temperatures. Summarily, rice Chl-deficient mutant gral was sensitive to temperature and showed more severe albino phenotype under low temperatures.(2) Through map-based cloning, we identified Os03g0255200 was GRA1 gene, which encoded an unknown function protein of DUF3353 family. Genetic complementation confirmed that Os03g0255200 corresponded to GRA1 gene. Protein structure prediction identified that the protein mainly included an N terminal transit peptide, a DnaJ domain and four transmembrane domains. Previous research identified that proteins with DnaJ domain usually interacted with Hsp70 proteins to perform its function. To identify the function of DnaJ domain in GRA1 protein, we performed yeast two-hybrid assay between GRA1 and Hsp70-7 and Hsp70-8 proteins which localized in chloroplast. The results showed that GRA1 protein can not interact with the two proteins. It indicated that the DnaJ domain is a DnaJ-like protein.(3) To research the expression pattern of GRA1 gene, we performed GUS staining and quantative real-time PCR (qRT-PCR) in roots, stems, leaves and spekletes tissues. The results showed that GRA1 expressed in all tissues. But it mainly expressed in leaves, especially in young leaves. Subcelluar localization identified that GRA1 protein was localized in chloroplast. Additionally, we performed qRT-PCR analysis for 11 genes associated with Chl biosynthesis in wild type and gral mutant under 20 and 30℃ temperatures. Compared to wild type, all of these genes were down-regulated in grcl mutant under 20℃ temperature. But under 30℃ temperature condition, most of these genes were no obvious difference between grcl and wild type. But the four genes HEMA, HEME2, CRD1 and DVR were up-regulated in mutant grcl. It indicated that the mutation of GRA1 gene also affected the expression of these genes.(4) Phylogenetic analysis showed that there were two orthologous gene of GRA1 in rice genome. One of them encoded a Chl-deficient gene YLC1. To understand the genetic relationship between GRA1 and YLC1 genes, we constructed the double mutant gral/ylcl.The double mutant seedlings of gral/ylcl showed albino leaves and died after the two-leaf stage under 20℃ and 30℃ conditions. The contents of chlorophyll a and chlorophyll b were zero in gral/ylcl leaves. Ultrastructural analysis of chloroplasts showed that the numbers of chloroplasts were less. And we also observed a hole in the chloroplast of the double mutant. The death reason of gral/ylcl should be that the development of chloroplast can not accomplish. Therefore, GRA1 and YLC1 genes were necessary to the development of chloroplast in rice.2. The main results of GRC1 gene are as follows:(1) Rice green-revertible chlorina 1 (grcl) mutant was isolated from a 60Co-irradiated mutant pool of indica rice cultivar Zhong 9B. And all agronomic traits were stable. Mutant grc1 showed a green-yellow phenotype before the four-leaf stage and gradually turned green. The levels of chlorophylls, carotenoids, and chlorophyll precursors were lower in grc1 mutant. Ultrastructural analysis of chloroplasts showed the number and size of chloroplast did not change obviously. The wild type contained grana thylakoid and starch granules, while grcl developed loosed thylakoid lamellar structures and less starch granules. Meanwhile, more osmiphilic bodies were observed in grcl mutant. The mutant had significantly lower plant height, number of tillers, and panicle length and headed significantly earlier than the wild type.In summary, the mutation of GRC1 gene not only affected the chlorophyll biosynthesis and chloroplast development but also the development of plant in rice.(2) Through map-based cloning, we identified a 45-bp insertion in the genomic region of Os06g0603000, which encoded a heme oxygenase 1(HO1). Expression of HOI was significantly down-regulated in the grcl mutant. We performed quantitative real-time PCR analysis of 11 genes associated with Chl biosynthesis in wild type and grcl mutant. Most of these genes were up-regulated. Pervious research showed that SE5 gene in rice and hyl Arabidposis, both encoding heme oxygenase, regulated oxidative stress response. SE5 RNAi transgenic plants showed decreasing transcripts of some representative antioxidative genes. To identify the responses of representative antioxidant enzyme genes to methyl viologen (MV), we induced the seedlings of WT and grcl within 8 h of exposure to 5 uM MV for real-time PCR analysis. Comparing to wild type, the expressions of SodAl and CatA were decreased in grcl, consistenting with pervious research. It indicated that HOI is GRC1 gene. Subcellular localization showed that GRC1 was localized in the chloroplast. In the research, we isolated and identified the gene for GRCl, which plays an important role in chlorophyll biosynthesis and chloroplast development in rice.