Establishment Of The Regeneration System Of Caragana Intermedia And Cloning And Expression Analysis Of Two PP2C

Caragana intermedia is a sandy shrub of drought resistance and cold resistance.It has a strong adaptability to the sand environment,and can be wind and sand to maintain soil and water and to improve local small environment.It has great significance to the protection and restoration of ecological balance.In the past two decades,the ecological environment in northwest China has deteriorated.Caragana is often planted as a major species in the process of vegetation restoration in sandy land.Breeding of Caragana intermedia with excellent genetic characteristics is an urgent as soon as possible.In the process of growing development,plant is difficult to escape a variety of volatile and destructive factors,such as low temperature,drought and high salt and other environmental factors.Plants form different physiological and biochemical mechanisms in order to adapt to the environment.According to report,PP2C participates in plant growth and development,cell cycle,signal transduction,osmotic stress and so on.So it is very important to study PP2C gene.In this study,we used the stem segments of C.intermedia as explants to produce regenerated plants by organ regeneration,and established the tissue culture system of C.intermedia.Explored the relationship between the main influencing factors and the culture effect,which lays the foundation for improving varieties and genetic transformation of C.intermedia;The CiPP2C8-like and CiPP2C27-like genes were cloned and analyzed by bioinformatics.The results showed that CiPP2C8-like gene was induced by salt and dehydration stress by RT-PCR.The main findings are as follows:1.Using the stem tips and segments of C.intermedia as explants to experiment on the callus induction.The results showed that the callus induction rate of stem segments was higher than that of stem tips.2.Using SPSS 19 to design four-factor three-level orthogonal test of GA3,6-BA,NAA and activated carbon AC.In MS medium of GA3 0.15mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.1g/L,the callus induction rate was 33.33%;The order of factors that affect callus induction was GA3,NAA,AC and 6-BA;The best hormone ratio is GA30.2mg/L+6-BA 0.2mg/L+NAA 0.4mg/L+AC 0.3g/L.3.In MS medium containing 6-BA(1.0mg/L)andNAA(0.2mg/L),the shoots induction rate was 13.33%.There are average 3-5 shoots per explant and are very healthy.In MS medium containing GA3(0.05mg/L),6-BA(0.05mg/L)and KT(1.0mg/L),the shoots induction rate was 20.00%,There are usually 1-2 shoots per explant and some of them are brown.4.Shoots growing to 2-3cm in length were removed and put in MS medium of GA3 0.15mg/L+IAA 0.5mg/L,the roots induction rate was 60%.Seedlings withroot were r were transferred into soil and the survival rate was 100%.5.The full length cDNA of CiPP2C8-like and CiPP2C27-like genes were cloned.The cDNA of CiPP2C8-like is 999bp,encoding a protein of 333 amino acids which was hydrophilic.The start codon is ATG and the stop codon is TAG.The cDNA of CiPP2C27-like is 1149bp,encoding a protein of 383 amino acids which was hydrophilic.The start codon is ATG and the stop codon is TAG..6.The expression of CiPP2C8-like and CiPP2C27-like was detected by Real-time-quantitative PCR.The results showed that the expression of CiPP2C8-like was up-regulated after salt treatment,and reached 33 times of that of the untreated samples at 48h.After the dehydration treatment,the expression of CiPP2C8-like was up-regulated and reached 252 times of the untreated at 12h.7.Construct expression vector CiPP2C8-like-HA.