Molecular Cloning And Expression Analysis Of 4CL Gene In Ziziphus Jujuba Mill.‘jinsixiaozao’and Z.jujuba Mill.‘wuhexiaozao’

Chinese jujube belongs to Ziziphus Mill.of the Rhamnaceae plants. Chinese jujube is a kind of important and specific fruit tree in our country, and it is the first great dried fruit in China. Z. jujuba Mill. ‘Wuhexiaozao’ is a rare and natural stoneless cultivar.The formation mechanism of stoneless hasn’t been found out. Except from stoneless, the characteristics such as branche, leave and fruit are the same basically between the Z. jujuba Mill.‘Jinsixiaozao’and ‘Wuhexiaozao’. ‘Jinsixiaozao’and ‘Wuhexiaozao’ were used as materials to study the formation mechanism of stoneless fruit in Chinese jujube. The 4CL（4-Coumarate:Coenzyme A Ligase） genes,which were related gene of lignification in the nuclear layer, were cloned from the fruit of ‘Jinsixiaozao’ and ‘Wuhexiaozao’ using reverse transcriptase PCR technology, and their expressions during the fruit development were studied. The main results were as follows:1. By sampling regularly to Chinese jujube seed-breeding base in Cangxian, Hebei, I took pictures as well as measured the vertical and horizontal, single fruit weight and fruit shape index.My analysis found that the ‘Jinsixiaozao’ was similar to ‘Wuhexiaozao’.2. A 4CL gene was cloned from the fruit of ‘Jinsixiaozao’ using reverse transcriptase PCR technology. This is the first time that a 4CL gene has been cloned from this species. The gene was 1820 bp long and contained a complete open reading frame of 1776 bp that encoded 591 amino acids（accession number KP893564）. The relative molecular mass of the protein was 63.726 KDa and its theoretical isoelectric point was 5.37. Through sequence alignment and structure domain analysis, we found that the 4CL gene contained several active sites, Amp binding sites, the COA binding sites of presumption and AAE consensus motif, at the same time contained Box I and Box II, which are conserved domains of the 4CL gene family. The gene is the new member of 4CL AFD- Class-I gene family and its secondary structure mainly is Random coil. Protein sequence alignment showed that the 4CL protein had the closest genetic relationship with Malus domestica protein XP₀08362825, and they belonged to the same evolutionary branch, the kinship was 82%.3.The c DNA sequence of ‘Wuhexiaozao’ 4CL gene was 1819 bp and contained a complete open reading frame（CDS） of 1776 bp that encoded 591 amino acids（accession number KP279930）. The relative molecular mass of the protein was 63.798 k Da and its theoretical isoelectric point was 5.31. Through the Ex PASy website analysis, 4CL gene of ‘Wuhexiaozao’ had multiple active sites, Amp binding sites, the COA binding sites of presumption and AAE consensus motif. As well as it included Box I and Box II of 4CL gene family. So it belonged to 4CL AFD-Class-I gene family. The secondary structure mainly was Random coil. Protein sequence alignment showed that it had the closest genetic relationship with Malus domestica protein（XP₀08362825）, the kinship was 82% and they belonged to the same evolutionary branch.4. Sequence alignment in NCBI, ‘Wuhexiaozao’and ‘Jinsixiaozao’sequence similarity was 99%.The physical and chemical properties, protein secondary and tertiary structure analysis showed that the similarity was very high, but they were different with 13 nucleotides and 6 amino acid residues.5.Used Bio Edit software,we searched 4CL gene from Chinese jujube genome.By comparison with CDS segment of 4CL gene,we found that we could not find 145 bp of CDS segment from the genome sequence. Used ‘Jinsixiaozao’ as the test materials, this segment genome sequence was obtained by gene cloning. After comparing we found that the genomic sequence of the 4CL gene contained 6 exons and 5 introns.6. Used Zj H3 gene as internal reference, by real-time fluorescent quantitative PCR technology the expressions of 4CL gene during the fruit development in‘Jinsixiaozao’and ‘Wuhexiaozao’ were studied. Results of q RT-PCR revealed that the 4CL gene in‘Jinsixiaozao’and ‘Wuhexiaozao’ was very different in the expression of different developmental stages. ‘Jinsixiaozao’ and ‘Wuhexiaozao’ were a trend of rising expression from the beginning of flowering, the expression of the 4CL gene peaked at about 30 days after flowering and decreased significantly from 40 days after flowering. ‘Wuhexiaozao’ expression was significantly lower than that of ‘Jinsixiaozao’. 4CL gene was an important factor in the process of stoneless formation.7.With ‘Jinsixiaozao’ and ‘Wuhexiaozao’ as templates,we got 4CL promoter. Used DNAMAN to compare the sequences, we found that the promoter sequences’ similarity was 99.64%, and there were only three different bases. The difference did not cause different promoter regulatory elements. Used Plant CARE analysis, we found that the promoter contained a lot of light responsive elements and lots of regulatory elements associated with hormone induced. The function of the other elements and specific regulatory elements also consisted in the promoter sequence.