Cloning And Primary Functional Analysis Of Lhd3 Gene Involved In Rice(Oryza Sativa L.) Heading Date

Heading date is one of the most important agronomic traits in rice, which determines the adaptability of rice cultivals in different areas and seasons. Rice is a model of short-day plant and its heading date is regulated by both the genetic and environmental signals. To improve the heading date of rice cultivals and break their limitation in rice breeding, it’s necessary to understand the mechanism of heading date genes.In this study, a chromosome segment substitution line DJY1-CSSL (Dianjingyou 1, a japonica variety in Yunan Province was taken as the recurrent parent, and O. meridional was used as the donor parent) was used, which shows 7.0 days earlier flowering than Dianjingyou 1 (DJY1) in Nanjing under natural long day condition (NLD) and 3.8 days later in HaiNan under short-day condition. Besides the SSR markers, a SNP detection array was also used to identify the genotypes of DJY1 and DJY1-CSSL. Several divergences distributed in 12 chromosomes were found between DJY1 and DJY1-CSSL, which means that DJY1-CSSL contains some exogenous segments. In order to establish the gene responsible for early heading date in DJY1-CSSL, a CSSL//DJY1 F2 segregation population derived from DJYl-CSSL and DJY1 was conducted for map-based gene cloning. The heading date of F2 population was continuous and ranged from 79 days to 96 days with a normal distribution. A preliminary linkage analysis was carried out by the F2 individuals with extreme early or late heading date and a co-segration trend near molecular marker RM1350 and RM8277 on chromosome 3 was found. Moreover, a major quantitive traits locus (QTL) was also detected in Marker RM1350 and RM8277, designated Lhd3(Late Heading Date on Chromosome 3), which can explain 23% of the phenotypic variation. In this QTL locus, a 3.4Mb segment was replaced and wherein a known heading date gene Hd6 was located and taken as one candidate gene. To establish the effect of this candidate gene, we cloned its cDNA both from DJY1 and DJY1-CSSL and sequenced. The results indicated that DJY1 has a dominate allele of Hd6 gene and encodes a functional protein. Compared with DJY1, a single base subsititution (A→T) was discoverd in DJYl-CSSL and produced a truncated protein. Based on the substitution, a CAPS marker Hd6-HindⅢ was designed and used to identify the genotypes of F2 indiduals with extreme early or late flowering time. Consistently, the substitution was found in all the individuals with extreme early flowering time, but not in any individual with extreme late heading date, which indicated that the single base substitution in Hd6 is responsible for the early heading in DJYl-CSSL.Lhd3 (Hd6) encodes a a subunit of CKⅡ protein in rice and delays heading date predominantly under long-day condition. Though this gene has been reported, the underlying mechanism was still elusive. For the purpose of the further investigation, functional analyses were conducted. Interestingly, two kinds of cDNA with different length were cloned and the intact one was 165 bp longer than the previously reported one, therefore encoding 55 animo acids more in the N-terminal. Subsequently, we screened the interacting proteins by yeast-two-hybrid and 11 proteins were selected. Among them, GRX (Glutaredoxin) plays a vital role in regulating rice heading date, thus, LHD3 may act by phosphorylating some subtrates, such like GRX. Moreover, we investigated the existence of A/T type in 42 rice varieties from independent resource. Only one indica c.v. Dhala Shaitta shows’T’type, the others show ’A’ type without exception, which suggested the T type is very few and has no significant correlation with heading date in these materials.