Construction Of Silencing Vector And Function-deletion Analysis Of Strawberry ARF4 Gene

The auxin response factor(ARF)is a class of transcription factors that regulate plant growth and development by regulating the expression of auxin-responsive genes.ARF4 is a member of the ARF family.The current research on ARF4 gene is mainly concentrated in plants such as Arabidopsis and tomato.There are few research on strawberry.The cellular localization of ARF4 protein is identified by constructing the ARF4-GFP vector.By constructing the strawberry ARF4 gene silencing vector,we aim to clarify the effect of ARF4 gene deletion on the growth and development of strawberry.The main results are as follows:1.First,the Fv ARF4 gene coding region sequence is amplified from the 'Ruegen' strawberry,which is 2409 bp in length and encod 802 amino acids.Its similarity with the corresponding sequence in the diploid forest strawberry genome is 99.79%;the amino acid sequence identity with the octoploid cultivated strawberry Fa ARF4 is 99%.The 333 bp specific sequence is selected by sequence analysis as a target fragment for constructing a silencing expression vector,and the positive and negative silencing fragments are amplified,respectively.2.The relative expression of ARF4 in different organs of forest strawberry 'Ruegen' is detected by SYBR dye-based q RT-PCR.It is found that the expression of ARF4 is highest in petiole,flower and leaf,followed by root and fruit.3.The 2400 bp Fa ARF4 sequence from which the terminator was removed was amplified using the full length sequence of the octaploid 'Yanli' strawberry Fa ARF4 coding region already in the laboratory.The ARF4-GFP fusion vector was successfully constructed by using p RI101-GFP as the backbone vector and Xba I/Sal I as the restriction site.Subcellular localization of ARF4 protein,which is found in the nucleus and belongs to nuclear protein.4.Using p ART27 as an expression vector and p KANNBAL vector as an intermediate vector,Xho I/Eco RI and Hind III/Xba I are used as restriction sites,and the 333 bp ARF4 positive and negative silencing fragment were inserted into p KANNBAL.Then,using Not I to perform single digestion,the sequence with ARF4 silenced fragment was ligated to p ART27 vector,and finally the strawberry ARF4 gene silencing vector was successfully constructed and named as p RNAi-ARF4.5.Using Agrobacterium-mediated 'leaf disc method' to infect strawberry leaves,and p RNAi-ARF4 vector is introduced into diploid forest strawberry 'Ruegen'.The relative expression of ARF4 in transgenic plants was quantitatively detected by SYBR dye-based q-PCR.ARF4 expression is significantly down-regulated in 6 transgenic plants.6.Through phenotypic observation,it was found that the leaf morphology of ARF4 gene-silenced plants did not change significantly,but showed late flowering and flower development defects: the petals couldnot be fully developed.Fruit development in transgenic plants was prone to malformation,and these phenotypes could be inherited into T1 plants.When observing the roots of T1 plants,it was found that the number of lateral roots of transgenic plants was significantly less than that of the control.7.By detecting the expression level of phenotype-related genes in transgenic plants,it is found that the expression levels of AP1,FT,SOC1 and SPL3 decrease,and the expression level of FUL and ARF3 do not change significantly.It is speculated that ARF4 may play a role by regulating the above genes.