Proteomics Of Kumquat Under Freezing Stress And MLP2-2 Gene Function Analysis

Kumquat(Fortunella swingle)is an evergreen fruit tree used as both medicine and eatables.It has high edible,health care,nutritional and medical value.In recent years,low temperature stress has seriously affected the development of the kumquat industry.The expression of the MLP2-2 gene would increase during the cold acclimation process of the kumquat in several studies.In this paper,the MLP2-2 gene was bioinformatics analyzed,extracted,transformed,expressed,and verified with different methods.At the same time,the different protein expressions between normal and cold acclimated kumquat were analyzed.The experimental results are as follows:1.The cold acclimation kumquat and normal kumquat were treated with cold stress,and the growth difference of kumquat under the two growth stages was compared.It was found that the cold acclimation kumquat can repair the frostbitten tissue and restore the growth state after being subjected to cold stress.Proteomics analysis of leaf protein found that compared with normal kumquat,cold acclimated kumquat increased the expression of protein related to substance synthesis,transportation and metabolism after cold stress,hydrolase,peptidase,etc.While the expression of hydrolase,peptidase related proteins were reduced.2.The bioinformatics analysis of the MLP2-2 protein revealed that the MLP2-2 protein has a signal peptide,and the secondary structure is composed of α helix,β sheet and random coils,and contains reaction site loops on the protein(reactive site loop),there is a transmembrane structure,and it is predicted that the MLP2-2 is an extracellular protein with13 phosphorylation sites,and has homology with the bean Kunitz protease inhibitor.As for the Bean Kunitz protease inhibitors can effectively inhibit protease activity,maintain cell homeostasis,and have certain resistance to abiotic stress.It is speculated that MLP2-2 gene may also have the same functional characteristics and play a similar role under cold stress.3.Two-year-old kumquat seedlings were subjected to cold acclimation,and the cds sequence of MLP2-2 gene was obtained by reverse transcription of leaf RNA,and the prokaryotic expression vector and transgenic Arabidopsis thaliana with MLP2-2 gene were constructed respectively.After transfered the p BRT7 plasmid to construct the p BRT7–MLP2–2 overexpression vector,and infected the Rosetta(DE3)Escherichia Coli engineering bacteria to obtain the target protein,the protein degradation rate of the Kumquat protein extract by the MLP2–2 protein were detected.The results showed that MLP2-2inhibited 16% of the degradation of kumquat protein,indicating that MLP2-2 protein has the function of protease inhibitor activity.In the comparison experiment of the phenotypes of transgenic Arabidopsis and wild-type Arabidopsis after cold stress,it was found that the leaves of transgenic Arabidopsis thaliana curled with water stains and were obviously frostbite under cold stress.However,after one week of recovery,the leaves stretched and the water stain was smaller,the MLP2-2 transgenic Arabidopsis thaliana repaired the frostbite tissue and avoided the occurrence of frost damage.This also indicates that the MLP2-2 gene is specifically expressed under cold stress,and produced a certain protein to resist the cold stress.