Phelipanche Aegyptiaca Strigolactone-sensing Gene PaMAX2 Clone And Its Function Study

Objective:In order to clarify the function of MAX2 gene,a key gene of strigolactone signal transmission,in the process of strigolactone recognition in Phelipanche aegyptiaca.To further understand the signaling mechanism of strigolactone,provide a theoretical basis for the use of strigolactone signal transmission pathway to prevent and control P.aegyptiaca.Method:The cloning of the strigolactone gene MAX2 by RT-PCR method was named PaMAX2.Bioinformatics software was used to predict the properties of the gene protein。Select specific fragments of PaMAX2 gene to construct a plant silencing vector with inverted repeat sequence and an overexpression vector of the gene,the regeneration system of P.aegyptiaca was established by tissue culture.The vector was transformed into P.aegyptiaca callus by Agrobacterium genetic transformation method to obtain RNAi plants and overexpression callus.Finally,count the parasitic rate of successfully transformed RNAi plants and overexpression plants,and use q PCR to detect the expression of the gene in transgenic plants,analyze the function of PaMAX2 gene.Results:1.The length of PaMAX2 gene was 2130 bp cloned into P.aegyptiaca by RT-PCR method.After comparison in NCBI,the sequence identity of the gene and the published P.aegyptiaca MAX2 gene reached 98.97%,Log in to Prot Param to analyze the physical and chemical properties of amino acids,the molecular formula of PaMAX2 is C₆₈₁₇H₁₁₃₆₈N₂₂₆₈O₂₈₁₇S₅₈₁,the molecular weight is 189.67 KD,the isoelectric point PI value is4.87,and it is weakly acidic.The protein was encoded with 754 amino acids,and the fat coefficient was 21.87,which is an unstable hydrophobin（instability index=44.21;GRAVY=0.744）.SPOMA online tool was used to predict the secondary structure of PaMAX2 protein.The prediction results showed that 333 amino acids formed the mostα-helix in the protein,accounting for 44.21%,282 amino acids formed random crimp,accounting for 39.83%,There are 86 extended chains,accounting for 12.15%.2.The seeds of P.aegyptiaca germination stimulated by GR₂₄ as explants,4 days after processing and use different media,different hormones and different sucrose concentrations to induce callus.The B5 medium was the most suitable medium for inducing flower buds,which consisted of B5+3%sucrose+600mg/L acid-hydrolyzed casein+0.7%AGAR.And we found that the sucrose content has a greater impact on the development of P.aegyptiaca,Under the same conditions,3%sucrose can induced more flower buds.At the same time,it was found in the process of tissue culture that P.aegyptiaca has mutual parasitism in the absence of the host.3.The callus of P.aegyptiaca was transformed into Agrobacterium tumefaciens,after antibiotic resistance screening and PCR molecular verification,transgenic callus was successfully obtained.Among the non-transgenic columns,using P.aegyptiaca in tissue culture flower bud state to inoculate melon（highly susceptible melon variety-Yifeng Naxigan）,the parasitic rate was 8%.The parasitism rate of callus decreased after Agrobacterium infection.This is because the parasitic ability of P.aegyptiaca callus itself is relatively weak,ater Agrobacterium infection and resistance screening,a few callus can continue to grow.After p XQ35-PaMAX2 RNAi-MSU440 was transferred to P.aegyptiaca.,the parasitic rate was lower than that of the control with Agrobacterium MSU440.Through the parasitism rate and PaMAX2 gene expression in p XQ35-PaMAX2 RNAi-MSU440 and p XQ35-PAMAX2-MSU440 plants,it can be analyzed that this gene plays an important role in the process of sensing the signal transmission of strigolactone.It affects whether the callus can perceive strigolactone,and it also has a certain impact on the parasitic rate.