Functional Identification Of Tomato Expansin Genes SlEXPA3 And SlEXLB1

Tomato(Solanum lycopersicum)is one of the most important vegetable crops in the world,rich in vitamin C and lycopene.Researches on tomato expansins are very early,but the biological functions of most of them are still unclear,due to the huge number of members of this family.In this research,two expansin genes,Sl EXPA3,which is fruit prefer-expressed and heat-induced,and Sl EXLB1,which can be intensively induce by ABA and drought stress,were selected for biological function identification.Both the over-expressing and gene edited lines of these two genes were obtained,and the characteristics of the Sl EXPA3-overexpressing transgenic lines(OEA3)were thoroughly studied,and the main results are as follows:1.Compared with the wild type,the OEA3 lines grown in greenhouse showed shortened internodes,increased internode number,tighter leaf arrangement,slightly higher chlorophyll content,and larger leaf angle,but no change was found in plant height and stem diameter.They have the same features in photosynthetic ability and resistance to Botrytis cinerea as wild type.In the field,OEA3 lines exhibited an increased internode number and an increased number and length of lateral branches before the first inflorescence bloomed.2.Compared with the wild type,OEA3 seedlings are more drought-tolerant,and indicated by some physiological features,such as relative water content,photosynthesis features,proline contents and SOD activity.3.In comparison to the wild type,the number of seeds in single fruit of OEA3 increased significantly,but there was no difference in their 1000-seed-weight.Spraying chemicals that can increase fruit-set rate on flowers did not lead to a difference in fruit setting rate between transgenic lines and wild types.Fruits on OEA3 lines ripened earlier than those of wild type.A concentration of 1-MCP that can severely postpone the ripeness of wild type fruits did not showed the same result on the fruits of OEA3 lines.4.A vector contained the 2 kb Sl EXPA3 promoter fragment and fused with GUS gene was constructed and were introduce to A57 through Agrobacterium-mediated transformation method.GUS staining of the transgenic lines showed that Sl EXPA3 was expressed in leaves,fruits and roots;5.Sl EXLB1 overexpressing transgenic lines exhibited no significant differences withwild type in plant height and stem diameter,as well as total soluble solid contents and hardness of fruits.GUS staining showed that Sl EXLB1 expressed both in leaves and roots;6.Site-specific editing vectors of both Sl EXPA3 and Sl EXLB1 were constructed independently using CRISPR-Cas9 system,and two Sl EXLB1 edited mutant plants were identified after molecular analysis.