Cloning Of PetSBP1 Gene Of Xinjiang Almond And Construction Of Yeast Two-hybrid Vector

Xinjiang wild,commonly known as dwarf almond belonging to the family Rosaceae and Prunus.is one of the oldest wild fruit trees in the world.At present,it is scarcely distributed in northern Xinjiang and Kazakhstan.Xinjiang wild almond belongs to the gametophyte self-incompatibility type.In this study,the focus is on the pollen characteristics of different populations of wild amygdalus in Xinjiang,define the change pattern of pollen vigor and stigma receptivity of wild amygdalus in Xinjiang,and then clone and obtain the key self-incompatibility gene SBP1.The detailed bioinformatics analysis will be performed,and the yeast two-hybrid vector will be constructed based on the SBP1 gene sequence.The results are as follows:1.Collect fresh flower pollen from Xinjiang wild almond plants,use pollen from five different natural populations of Xinjiang wild almond,and adopt I2-KI staining and pollen digression to complete the testing of the daily changes and weekly changes of pollen viability of different populations of Xinjiang wild almond Determination of changes.Employ different concentrations of pollen medium formulations to screen the optimal medium components of Xinjiang wild almond The test results found that the vitality of the five different populations of wild Almond in Xinjiang reached the strongest at 10-12o’clock in the morning.In terms of weekly changes,the pollen activity of the five populations of Xinjiang wild Almond reached the maximum on the fourth day after flowering.After selecting the different components of the pollen medium for the optimal pollen medium.it was found that the best pollen medium formula of Xinjiang wild almond is 1%agar,10g/100 ml sucrose,0.05 g/100 ml boric acid,0.009g/100ml calcium chloride.The optimal culture temperature is 25℃ and the culture time is 8h.2.The RNA extraction from collected Xinjiang wild amygdalus pollen and the cloning and sequencing of the SBP1 gene showed that Xinjiang wild amygdalus SBP1 gene was 1211 bp in total length,its maximum open reading frame was 1017 bp.and G+C content was 44.43%,A+Tcontent was 55.66%,encoding a total of 338 amino acids.The molecular formula of the encoded protein is C1648H2618N470O529S25,the theoretical isoelectric point is 5.29,48 negatively charged residues(Asp+Glu),36 positively charged residues(Arg+Lys).fat coefficient 73.85,and instability index 54.49 With an average hydrophobicity index of-0.584.PetSBP1 is a water-soluble labile protein.Carry out analyzing of potential phosphorylation site of amino acid sequences The encoded amino acid sequence has a close relationship with sweet cherries,and farther with potato and Arabidopsis.3.The obtained Xinjiang wild almond self-incompatibility key gene SBP1 was specifically cloned into the vector pGADT7,and the bait vector pGADT7-SBP1 which can be used for yeast two-hybrid was obtained.The obtained induction vector was sequenced and self-activated to find out that the constructed yeast double-hybrid bait vector had no self-activation,and the transformation product was non-toxic.The sequencing results showed no frameshift mutations and mismatches,indicating that the Xinjiang wild almond self-incompatible and non-incompatible pGADT7-SBP1 yeast double-hybrid vector can be used for yeast double-hybrid.The follow-up work of the hybridization system laid the foundation for further screening of proteins interacting with SBP1 from Xinjiang wild almond self-incompatibility cDNA library and for exploring the molecular mechanism of self-incompatibility in wild almond trees in Xinjiang...