Gene Map-based Cloning Of A Rice Dull Mutant W54 And Phenotypic Analysis And Gene Mapping Of A Thermo-sensitive Yellow Leaf Mutant Yl2(t)

Screening of rice mutant is an important approach to study the process of rice development and regulation mechanisms.Map-based clonings of rice mutant genes and functional analysis of the mutant genes related are of great significance for understanding metabolism pathway and identifying new factors in rice.In this study,the mutant gene of the rice dull mutant w54 was map-based cloned based on the results of Wang Peng.Meanwhile,the expression levels and activities of starch biosynthetic enzymes were analyzed in w54 and wild type endosperms.In addition,a thermo-sensitive yellow leaf mutant yl2(t)was isolated from the M2 population of MNU treatment of japanica cv.Koshihikari.The phenotype of yl2(t)was analyzed and the mutant gene was primarily mapped.The main results are as follows:1.The w54 mutant exhibited dull endosperm with a significantly decreased amylose content.The semi-thin splice showed that compound starch granules in w54 endosperm developed normally and were lightly stained by I2-KI compared with those of wild type.2.The accumulation curve of storage substances showed that the w54 mutant displayed a significantly slower rate in storage substances accumulation than wild type at each measurment stage except 15 days after fertilization(DAF).The accumulation rate of amylose content in w54 was significantly lower than that in wild type at the same time.3.The mutant w54 gene was previously mapped to a region of about 280kb on the long arm of Chromosome 6.The w54 mutant gene was finally mapped to a region of about 187kb between markers W72 and W47 on Chromosome 6 using a larger mapping population of 900 recessive individuals and new molecular markers.This region contains 26 open reading frameworks and a replacement of C to T was found in eighth exon in the twentith gene,leading to a premature stop codon.4.qRT-PCR showed that the expression level of Wxb gene was significantly decreased in w54 endosperms compared with wild type at 6 and 9 DAF,especially at 9 DAF(only one tenth of WT),but recovered to normal level at 12 DAF.Semi-quantitative PCR showed that the w54 endosperm at 12 DAF exhibited a higher level of abnormal Wxb mRNA than the wild type endosperm.5.Expression levels of BE ?a,Susy and SS ?b were found significantly decreased in the w54 endosperm at 12DAF compared with wild type via qRT-PCR assays-however,other genes showed little significance.6.Western blotting analysis showed that no changes in the contents of ADPG pyrophos-phorylase 2b,ADPG pyrophosphorylase L2,plastidial phosphorylase 1,starch branching enzyme I and starch synthase ?a were noted in w54 endosperm at 12DAF.7.Native-PAGE and activity staining analysis showed that the activities of isoamylase,pullulanase,starch synthases I and starch phosphorylase displayed no significant differences between w54 and wild-type,while the activity of starch synthase?a was slightly increased in w54 endosperm at 12 DAFTaken together,compared with wild type,the expression level of Wxb was significantly lower in w54 endosperm but abnormal splicing product of Wxb pre-mRNA increased.We speculated that W54 protein probably participate in amylose biosynthesis through transcriptional regulation or post-transcriptional regulation of Wxb.8.The yellow leaf mutant yl2(t)seedlings showed yellow leaf and significantly lower chlorophyll contents with unaffected development of chloroplasts when growing under continuous 20? for 15 days and was lethal if extending time of low temperature treatment to 20 days,but turned to normal green color when transferred to 30? or growing under continuous 30?.However,agronomic traits of yl2(t)were not affected by the mutation in the filed.qRT-PCR showed that the expression levels of genes involved in chlorophyll synthesis in yl2(t)seedlings had different changes under the two temperatures.Genetic analysis suggested that the yellow leaf phenotype of yl2(t)was controlled by a pair of recessive genes and finally the yl2(t)gene was mapped to a 647kb region between the markers N8-36 and P8 at the terminus of long arm of Chromosome 8.YL2(T)is a new gene controlling chlorophyll synthesis in rice.