Construction And Analysis Of Potato Enhancer-trapping Lines

Potato is not only fourth largest food crop in the world,but also is fourth major grain crops in china,and it has played a more and more important role in people's life.After the completion of the whole genome sequencing of potato,it helps people to deepen understanding of the genetic law of the potato and to strengthen the development of its rich genetic resources,at the same time,it helps to accelerate the use of molecular breeding.Double haploid potato(DM DM13-516-R44)is one of the sequencing material.This experiment used DM as material,through Agrobacterium mediated guiding,established completing enhancer trapping T-DNA vector into potato explants and obtained the transgenic plants,and then screened and identified the positive transgenic plants.Initial constructed the potato enhancer capture line population,and carry on the molecular identification.We have established a set of suitable for amplification of flanking sequences of the mutant plants PCR-walking technology system,and analyzed it according to the published sequence of potato.The main experimental results are:1.This research initial established the potato enhancer-trapping lines,and obtained about 12000 transformed seedlings plants.we carried out 1500 transformed plants' positive identification and gained 978 positive lines.At the same time,we optimizated the transformation system of potato DM,the main experimental conditions are: Differentiation culture medium Cephalosporium hormone(CEF)concentration is 600mg/L,Hygromycin hormone(hyg)is 8mg/L,Explant pre-culture for 13 days,Infection time was 8min,Agrobacterium concentration OD600=0.4,Co-culture 3 days.2.This research uses PCR-walking method amplified potato mutants flanking sequences of system and amplified 60 positive mutants' flanking sequences.Sequenced 28 mutants and finded that there are 6 mutants lines is to insert valid and analysised the insertion site informations preliminarily3.This research obtained 30 mutants strains of micro seed potatos and observed the phenotypic changes of some mutants.460 positive mutants were identified and their phenotypic changes were observed.