Clone And Analysis Of The Genes Related To Photoperiod Sensitivity In Maize

Tropical and subtropical germplasms contain abundent hereditary variation to broaden Chinese maize germplasm and increase the breeding level.However,photoperiod sensitivity is the main restriction of its use in temperate zone.We have to study it in order to utilize tropical and subtropical germplasm better.CML288,a tropical maize photoperiodic sensitivity inbred line,and HuangZao No.4,a temperate maize inbred line were used as material in these studies.We studied the responses to photoperiod further in CML288 and cloned some photoperiod-related genes,ZmHd1,PL5L15 and PL3K2,which play the important roles during the transition from vegetative growth to reproductive growth,and test its transcription level by Real-Time PCR during different development leaf number stage and a whole day in photoperiod sensitivity stage.The ZmHd1 gene was mapped and deduced its function by comparing with the QTLs result of photoperiod sensitivity RILs.The main results were as follows. The inbred lines were grown in 2 different controlled photoperiod recycles,9hr and 15 hr light every day.Result showed that the HuangZao No.4 lines could perform productive growth and elongate the stem not only short-days conditon but also long-day condition.While CML288 could not development tassel and elongate the stem on long-day condition.CML288 was much more sensitive to photoperiod than HuangZao No.4.It shows that the tassel difference transferred from SD to LD condition in sixth leaf period was invisible but in seventh,the tassel development between seven-leaf and eight-leaf period were later than the tassel in short days consistently.The result indicated that CML288 was more sensitive to photoperiod between sixth and seventh leaf period.CDS and DNA sequence of ZmHd1 were isolated and cloned successfully using RACE technique.The full-length CDS sequence was 1310 bp,include a 1197bp complete ORF region,which encode a 397 amino acid peptides,owning two highly conserved BBOX and CCT domains.ZmHd1 genome gene was composed of one intron and two extron,had complete transcript elements,such as promoters and terminal sequence.It is 74 percent identifies,and 75 percent positives with Hd1 of Oryza sativa.It is an ortholog of Hd1 and related to photoperiod sensitivity. We found a premature stop codon in the second exon of ZmHd1 of Huangzao4 due to a single basepair replacement from T to A,which is not found in the same position in CML288.The translation of ZmHd1 protein had to stop ahead,and the conserved CCT domain in CO/Hd1 family was lost.So it may be an important mechanism of the change of photoperid sensitivity between tropical and temperate lines.We designed to develop a CAPS marker that depend on the difference of StuI restriction site caused by the premature stop codon to detect the photoperid sensitive gene in maize. We found the ZmHd1 gene sequence shared a high similarity with the sequence of AC189046 in Maize genome database(MaizeGDB),and it was mapped in the chromosome 9.03,near centromere.We detected a QTL in chromosome 9.03,which is benefit to photoperiod sensitivity.So it considered that ZmHd1 was a candidate gene of this QTL region.We studied the expression of ZmHd1 by Real-Time PCR, the result showed that it has the uptrend in photoperid sensitive time,and expressed higher level in long day condition than in short day condition.A subtracted,cDNA library was constructed by the way of suppression substractive hybridization with the mRNA of leaf and shoot apical meristem of 7^th leaf stage,it contained more than 3000 clones related to photoperid sensitivity.We analyzed 64 clones,results showed that 16 EST sequences were high homologous with maize sequences related to transcription factors,signal transduction,development,flowering, adversity-stressed,and so on in GenBank.31 EST sequences were homologous with other plant genes.7 EST sequences may be the new genes.We cloned the PL5L15 gene after SSH cDNA library construction.The complete sequence was 1410bp,a predicated 897 bp open reading frame was detected in the cloned sequence,which encoded 292 amino acids,a conserved Clp-protease domain was found between 89^th and 272^nd amino acids.It was positive 78 percent with ClpR2. The RT-PCR results showed that PL5L15 is a ClpR2-LIKE gene,related to reproductive definition in photoperiodism.We cloned the PL3K2 gene based on the EST from SSH cDNA library.The complete sequence was 639bp,a deduced.453-bp open reading frame was detected in the cloned sequence,which encoded 292 amino acids,a conserved Jaclin domain was found among the amino acids.The RT-PCR results shows that P L3K2 is a subfamily gene of Letins in Maize,related to resistent response in photoperiodism.