The Study On The Tissue Culture Of Loquat And Bioinformatics Analysis On Its Sucrose Synthase (SS) Gene

Loquat is a characteristics fruit in the south of China. Until now, breeding new varieties mainlyrely on the traditional methods which mean long time and heavy workload but limit the use of manybiotechnology methods in related researchs. Thus, find out a new way to change the molecular so thatthe cultivar of Loquat will be better is necessary. while establishing a stable regeneration system bytissues culture and finding out the gene with specific function will be the pre-work for the implement oftransgenic technology.This article mainly use the material like leaf and embryo of “Ninghaibai” loquat to explore theinfluence of culture medium formula, disinfection method, light and temperature on the loquat tissueculture, hope to provide a basis for the establishment of a stable loquat regeneration system of loquat;Meanwhile doing the SS gene sequencing bioinformatics analysis obtain from RNA-seq by usinganalysis software such as NCBI, DNAMAN, MEGA5.0ect,, to lay the foundation of the research ofloquat sugar metabolism related genes and the further research of fruit quality formation mechanism;Ultimately lay the foundation for genetic transformation system to provide new way for loquatimprovement and breeding new varieties. Here are the results:1. Use10%NaClO to sterilization step by step for15min can effectively reduce the browning andpollution rate. In different light treatment experiment,20d dark pretreatment could improve the leafcallus’s quality. But it seems no obvious effect to the formation of mature cotyledon callus. Embryosseeds will turn green from dark to light.2. Medium mature leaf does better within different maturity leaves in leaf callus induction. And2,4-D plays an important role in it. The optimum culture medium for leaf tissues is MS+0.5mg/L2,4-D+0.5mg/L6-BA. And the induction rate is84.2%. The optimum temperature is25±2℃, Too high canlead to tissue browing.3. Use the cotyledons as explants with different cutting method in the experiment, we found that itcan induce some green compact callus with granular protrusions in proper conditions. The transversecotyledon with good germ can germination in nature way，while its wound can induce less callus. Theoptimum culture temperature is25±2℃. MS+0.8mg/L2,4-D+0.4mg/L6-BA+0.4mg/L NAA is thebest medium for embryo callus culture with86.8%induction rate, and it also works in leaf callusformations. 4. A certain concentration of TDZ and NAA could give birth to cluster bud. MS+1.0mg/L TDZ+0.4mg/L NAA could directly stimulate the explants including the cotyledon with germ, the branch ofseedling induce bud,5per explant. But it’s difficult to induce adventitious bud through somaticembryogenesis, only some kinds of abnormal organ have been obtained. It may attribute to theimmature of somatic embryogenesis and unsuitable culture medium.5. Using analysis software such as NCBI, DNAMAN, MEGA5.0to do the sequencingbioinformatics analysis in SS gene which obtain from RNA–seq. There are6different SS genesequence with high homology which can be95%, each of them contains an open reading frame whichcan encode807amino acids. Protein analysis shows that they are hydrophilic protein withouttransmembrane or signal peptide. Alpha helix is their main structure. Conserved protein domain predictsit has four ADP binding sites and two conservative domain, sucrose synthetase functional areas andglucosyltransferase functional areas. Phylogenetic analysis shows that SS gene of Eriobotrya japonica L.has high homology with prunus persica, citrus ect.