| In China, the cultivation of litchi (Litchi chinensis Sonn.) has continued for about 2,000 years at least. According to the maturity period, litchi variety was divided into early-maturing, middle-maturing and late-maturing variety, and in the long process of artificial cultivation and natural selection, there were a wide range of varieties and types formation. Furthermore through the introduction, the late-maturing litchi germplasm resources in many areas have been under consistent using. The late-maturing litchi genetic map consisting of excellent gene was the material basis of genetic improvement of litchi. In view of this, 30 accessions of late-maturing litchi germplasm gathered in Fuqing City (Fujian) and other places were used to perform analysis of genetic polymorphism using RAPD analysis techniques to select the core collection of late-maturing litchi germplasm. On this basis, embryonic callus induced from anther was cultured by restricting growth conservation method, and GRX gene was cloned from embryogenic callus of litchi, which laid a foundation for further study of in vitro conservation. The main findings are as follows:1. RAPD analysis on late-maturing litchi germplasm resources30 accessions of late-maturing litchi germplasm were used to perform analysis of genetic polymorphism using RAPD analysis techniques. The results showed that: 277 RAPD bands were amplified by polymerase chain reaction with 19 random primers, and 92.06% of which were polymorphic. According to the results of RAPD analysis, 30 accessions of late-maturing litchi were determined using Jaccard cluster analysis, and the results showed that: the genetic similarity coefficient among them varied from 0.427 to 0.942 and the average genetic similarity coefficient was 0.625, and that according to the results, 30 accessions of late-maturing litchi germplasm was reassigned into two classes by D1=19.0, and which could be divided into six subgroups by D2=15.0, and 24 groups by D3=5.0. Litchi variety of the same origin could be basicaly classified into a class, but some could not, which suggested that some had relatively distant genetic relationship with other species and there was some variation. Comprehensive consideration, the least amount of resources and large genetic differences and other factors, there were 20 litchi varieties identified as the core collection:'Yuanhong','Xiafanzhi','Haiken4','Edanli','Zili','Xiushili','Jiangkouli','Niuxinli', varieties of'Niuxinli','Dadingxiang','Nandaowuheli','Chuntengmili','Hongxiuqiu','Dongguanwuheli','Yurongwanli','Maguili','Shishengshu', 'Meiguixiangli','Liqiuli'and'QingA13'.2. Studies on restricting growth conservation and induction of embryonic callus from anther of late-maturing litchiEmbryonic callus was induced from anther of the core collection of 20 late-maturing litchi. The results showed that different kinds of varieties had different induction rates, and the average induction rate was between 1.7050% and 63.3233%; and that different media of the same species also had different callus induction rate of anther. Moreover, the best medium for in vitro conservation was determined:'Yuanhong': 1/4MS +1.0 mg·L-12,4-D +25 g·L-1mannitol +25 mg·L-1PP333 +0.4 g·L-1betaine;'Haiken4': 1/2MS +1.0 mg·L-12,4-D +20 g·L-1mannitol +25 mg·L-1PP333 +0.1 g·L-1betaine, with 20 g·L-1sucrose and 7 g·L-1agar, pH 5.8, 25±1℃, cultured in dark, which could prolong the sub-culture cycle of embryogenic callus induced from the anther of late maturing litchi to 140 d.3. Cloning and bioinformatics analysis on glutaredoxin in embryogenic callus from late maturing litchiCombined homology cloning with RACE, the embryogenic callus iduced from late-maturing litchi'Xiafanzhi'was used as material to cloning the resistant gene GRX. The full-length cDNA sequence of GRX was 697 bp, and 5'UTR was 81 bp, while 3'UTR was 217 bp. The region of GRX also contained a typical polyadenylation signal AATAA and 3 'end also contained 23bp poly (A). The sequence of GRX had high homology with the sequence of GRX in other plants having been logined on GenBank. And the assembled sequence contained an open reading frame of 366 bp encoding 132 amino acids, with ATG as start codon and TGA as stop codon. Accession number of GRX in GenBank was GU250736; and after logining on Genbank, bioinformatics analysis of amino acid sequence of GRX showed that relative molecular weight of glutaredoxin was 14.6067kDa, and the isoelectric point (pI) was 6.41; Furthermore, GRX was a hydrophilic protein, which contained transmembrane domain; and the maximum potential that its sub-cellular localization was in the mitochondrial membrane was 0.850. The possibility of GRX with the signal peptide and belonging to secreted proteins were up to 99.9%. Functional domain analysis of conserved domains of GRX, in late maturing embryogenic callus, showed that there was an active center, whose amino acid motifs was CPYC. By function prediction and analysis, it provided evidence that glutaredoxin gene involved in the scavenging process of oxygen free radical in vitro conservation.
|
PDF Full Text Request