Functional Identification Of Stress Resistance Of Phenyla Lanine Ammonia-lyase Gene(PAL)in Astragalus Membranaceus

There is still a shortage of landscaping plants in cold,arid areas,Genetic engineering breeding of resistant plants is one of effective and quick ways to enrich the plants under adverse environmental conditions.Among them,it is particularly important to screen the functional genes resistant to various abiotic stresses.Astragalus membranaceus is an ornamental and medicinal plant which has high resistance characteristics.It is extensively applied in cold,arid and saline areas.And it is also an important resource for stress-resistant plant genes.The phenylalanine ammonia-lyase（PAL）is widely exist in plant.As a bridge between the secondary and primary metabolism of phenylpropane,PAL usually plays an important role in physiological metabolism and stress response of plants.In this study,the expression characteristics of the cloned astragalus AmPAL gene under adverse conditions were analyzed,the subcellular localization and the expression stability of the progeny of AmPAL transgenic tobacco were tested,and the effects on the morphological development of tobacco were investigated.The antistress function of AmPAL gene under various abiotic stress was analyzed.The protein interaction network was constructed of NtPAL1 in tobacco with high homology were used to further explore the PAL resistance mechanism,Meanwhile,the allogenic transformation of AmPAL gene in ornamental flower lily and petunia was carried out to improve the stress resistance of flowers.This achievement can provide gene resources for the development of anti-stress gene engineering and breeding of flowers in the future,and also lay a theoretical foundation for the research on anti-stress mechanism and development and utilization of astragalus.Below are key research findings:1.The full-length cDNA sequence of the PAL gene of astragalus was obtained by RT-PCR（AmPAL,EF567076）,which has the Lyase_aromatic domain and belongs to the phenylalanine ammol/lonia-lyase family.NJ phyletic tree analysis found that PAL of astragalus astragalus was aggregated with pea（Pisum sativum）,soybean（Glycine Max）and Medicago sati of the same family,and the similarity was 87.59%,89.18%and 57.36%,respectively.2.The AmPAL is a functional gene in response to salt,alkali and drought stress and ABA signal induction.The expression of AmPAL was significantly increased in roots and leaves under saline（NaCl）,alkali（NaHCO3）,PEG6000 and ABA stress conditions.Especially in the roots,,AmPAL was up to 9.5931-fold higher after 6h under saline stress.For alkali stress,AmPAL was expressed up to 24.5243-fold higher at 48 h.In the leaves,PEG6000 stress induced the expression of AmPAL gene significantly increased,until 48h,the stress was still increasing.And the roots increased first and then decreased.But were higher than the control.The ABA signal was induced the peak of expression at 24h.The amount of induced expression in the roots is also increasing trend.3.The expression vector pBII21-AmPAL-GFP was constructed and transferred into onion epidermal cells by gene gun method.The green fluorescence of ampal-gfp fusion protein was detected,indicating that AmPAL subcells were located on the cell membrane.4.The plant expression vector pCXSN-AmPAL was constructed and the vector was transformed into tobacco.The overexpression of AmPAL in T3 generation tobacco was detected by Southern hybridization and Northern blot hybridization.5.Under stress conditions,the AmPAL gene has a series of physiological resistance reactions and shows resistant growth.The Plant resistance was enhanced By reducing membrane lipid oxidation damage and improving photosynthetic efficiency Under stress conditions.Studies on the correlation between the T3 generation of AmPAL-overexpressing tobacco and stress resistance showed that leaf and root growth of transgenic tobacco with AmPAL in two-leaf age seedlings were inhibited under NaCl stress,but root length growth,fresh weight and PAL enzyme activity were better than WT.Na+/K+ of transgenic tobacco was significantly higher than WT,which improved the utilization rate of K+and showed growth advantage under stress.Under NaHC03 stress,which caused changes in the pH value of cultivated substrate,the tobacco roots of AmPAL gene were longer than WT,but MDA was lower.The growth of transgenic tobacco seedlings with 5 leaf age was inhibited with the increase of treatment concentration under saline,alkali and drought treatment,but the overexpressed AmPAL tobacco showed growth advantages.Under the same concentration treatment,the SOD activity,proline content,chlorophyll content and chlorophyll fluorescence parameters of overexpressed AmPAL tobacco seedlings were detected to be superior to WT.6.AmPAL may participate in the growth and development of plants in some way.Through the observation of the growth and development of over-expressed AmPAL T3 generation nicotiana tabacum,it was found that the plant was shorter,the pitch spacing was shortened,the ground diameter was larger,the leaves were wider,the ratio of leaf length to leaf width was smaller.The flowering period was postponed for 52d,and the number of flowers increased and the flowering period also increased.7.The homology of AmPAL and NtPAL gene by multiple sequence alignment in NCBI BLAST,Test results show that AmPAL and NtPAL1 had the highest homology,the results showed that the gene promoter contains a lot of adversity response components and optical response of the related components,Under adverse conditions,these stress response elements will activate the expression of NtPAL gene.NtPAL1 protein interaction network analysis found 10 proteins,XP₀09622435.1,XP₀09612485.1,XP₀09631403.1,XP₀09631897.1,XP₀09609722.1 and XP₀09607087.1 participates in the metabolic pathway of phenylalanine,XP₀09612485.1 and XP₀09631403.1 participates in the synthesis of betaine,XP₀09622435.1、XP₀09631897.1.XP₀09612485.1 and XP₀09631403.1 are Basic types and Forms of vitamin B6,which are all metabolites to improve plant resistance.It fully shows that PAL is the key gene to improve the antistress function of plants.8.The pCXSN-AmPAL plant expression vector was constructed,and the AmPAL gene was transferred into Petunia hybrida and Lilium Pumilum by Agrobacterium-mediated method.Through the resistance screening and PCR detection of resistant plants,it was proved that the target gene has been successfully inserted into the genome of Petunia hybrida and Lilium Pumilum.Overexpression of AmPAL in transgenic leaves of Lilium Pumilum was detected by real-time PCR.It can be considered that AmPAL gene has stress-related function.Under stress conditions,there was an interaction between AmPAL gene stress resistance and membrane lipid oxidation resistance.It is speculated that AmPAL gene may be involved in ion transport in plant cells.