Mechanisms Of Rapid Growth And Stress Tolerance In Arabidopsis Pumila,a Typical Ephemeral Plant In Xinjiang Desert Environment

In recent years,climate change has led to an increase in extreme weather,and the growing global population has brought unprecedented challenges to agricultural production.Crop genetic improvement can promote food and nutrition security,which has become one of the important issues concerned by scientists.However,the current crop breeding strategy lacks sufficient efficiency,and it is difficult to meet the short-term or long-term food production needs.It is necessary to combine traditional breeding,modern biotechnology,genomics research and“speed breeding”to accelerate the process of crop improvement,which help us meet the challenge of food demand of 10 billion people.Genomics can maximize the effective utilization,diversity,grain yield and security of resources,but it is necessary to identify as many germplasm resources as possible at the level of genome and agronomic traits,so as to explore and identify more excellent gene resources for crop genetic improvement in the future.Marginal land is a strategic reserve cultivated land resource to ensure China's food security.There are30.752 million mu of marginal land in Xinjiang,and saline alkali,Sandy and drought are the main obstacles.Ephemeral plants are widely distributed in the margiinal of Xinjiang,China.They play important roles in preventing wind and fixing sand,conserving soil and water,improving micro habitat and protecting surrounding farmland from sand damage.Therefore,they have made important contributions to the protection of agricultural biology and ecological environment especially in early spring in Xinjiang.Study of the molecular mechanism of ephemeral plants adaptation to the environment will provide scientific basis and theoretical support for better utilization and protection of ephemeral plant resources,which is of great significance for future crop breeding and development and utilization of marginal lands.Arabidopsis pumila,a cruciferous plant growing in the desert area on the southern edge of Gurbantunggut Desert,is characterized by rapid flowering and fruit bearing,large fruiting capacity,high photosynthetic efficiency,salt tolerance,and cold resistance,which contains rich resistance gene resources.In this study,we used A.pumila as the research material,established biological research systems from the physiological and cellular level,molecular biology aspect,combined with RNA-seq technology to explore the mechanism of its adaptation to special habitats and analyze the value of resistance gene mining and breeding.The main research contents and results are as follows:1.Establishment of tissue culture and genetic transformation system of A.pumilaFirstly,the tissue culture experiment was carried out,using tender roots,hypocotyls,leaves and petioles as explants.The callus and multiple adventitious buds were effectively induced on the induction medium of MS supplemented with 0.5 mg/L 6-benzylaminoadenine and 0.1 mg/L naphthylacetic acid.On the same medium,young roots,hypocotyls,leaves,and petioles could induce callus,but the rate of young roots was the highest.Furthermore,the leaf and petiole of 4-week-old seedlings were used as explants,the?1-pyrroline-5-carboxylate synthetase 1(P5CS1)gene of A.pumila was used as the target gene,hygromycin B was used as the screening antibiotic,and the explant pre-culture,infection and co-culture were used to study the genetic transformation method mediated by Agrobacterium tumefacien GV3101.The results showed that the callus and adventitious buds could be induced from the four explants of young root,hypocotyl,leaf,and petiole.The callus induction rate of root explant was the highest,while that of hypocotyl was the lowest.The results demonstrated that the most suitable concentration and time of Agrobacterium infection were different,but the callus and adventitious bud could be formed under the suitable concentration of Agrobacterium infection.Further using inflorescence infection method to infect the inflorescence of A.pumila,it was found that the transformation efficiency of Agrobacterium could be improved by appropriately prolonging the infection time within a certain time range.When the infection time was 30 s,the screening efficiency could reach 0.67%.2.Screening suitable internal reference genesThe appropriate internal reference genes are the important prerequisite for the accurate analysis of gene expression using quantitative real-time PCR(qRT-PCR).In this study,10 candidate genes,including ACT1,ACT2,ALDH,EF1B,GAPDH,HAF1,LOS1,UBC35,UBQ9,and UEP;the KUP9,encoding a potassium ion absorption osmosis enzyme,is selected as the verification gene.Four abiotic stresses(drought,heat,cold,and salt),were applied to A.pumila.Samples were collected at 0,3,6,12,24,and 48 h,including seven different tissues:root,hypocotyl,cotyledon,rosette leaf,stem,flower and siliques.RNA was extracted from the samples for qRT-PCR assays.The expression stability of 10 internal reference genes was analyzed using ge Norm,Norm Finder,Best Keeper,and Refinder softwares.The results were as follows:(1)ACT1 and GAPDH were the appropriate internal reference genes in different tissues;(2)HAF1 and UEP were the appropriate internal reference genes under 10%PEG6000 stress;(3)GAPDH and ACT1were the appropriate internal reference genes under 250 m M Na Cl stress;(4)GAPDH and UBC35 were the appropriate internal reference genes under low temperature(4?)stress;and(5)Under high temperature(40?)stress,the suitable internal reference genes were GAPDH and UBQ9.On the whole,GAPDH and UBQ9 are the most suitable combination of internal reference genes in all samples.The expression patterns of the KUP9 gene further verified the stability of the selected internal reference gene,indicating that the selected internal reference gene is suitable for the standardization of gene expression.3.RNA-seq analysis of different growth and development stagesAfter germination in early spring,A.pumila grows rapidly.The phenotypic changes of A.pumila during the whole growth cycle in natural habitat were analyzed.The results showed that the plant height and leaf number increased most significantly in one month after germination,especially in the five stages of bolting(grow stage,GS1),flowering(GS2),podding stage 1(GS3),podding stage 2(GS4),and podding stage 3(GS5).The morphological changes from vegetative growth to flowering and fruiting,as well as pod growth were observed.The leaves of GS1,GS2,GS3,GS4 and GS5 were selected for RNA-seq analysis.Fifteen libraries were constructed,and 694,576,138 raw reads and 660,395,266 clean reads were generated after sequencing,with a total sequencing amount of 99.06 G.Correlation coefficient and principal component analysis showed that there was no significant difference between GS1 and GS2,and the number of differential genes was the least,and the difference between the other groups was very significant.A total of 29,994 differentially expressed genes were found in five developmental stages.Go and KEGG enrichment analysis showed that most of the differentially expressed genes were enriched in photosynthesis,ribosome,physiological rhythm,?-linolenic acid metabolism,oxidative phosphorylation,flavonoid biosynthesis,and glucosinolate biosynthesis.Compared with GS3 and GS2,GS4 and GS3,GS4 and GS4,the number of differentially expressed genes related to physiological rhythm was 24,27,and 24,respectively;the 15 genes related to photoperiod flowering pathway such as CO,GI,and FT were significantly differentially expressed.GO enrichment analysis revealed that the KT/HAK/KUP gene family was significantly enriched in the whole process of pod setting stage,suggesting that they play cruical roles in the regulation of the growth and development of A.pumila.4.Genome-wide identification and analysis of the KT/HAK/KUP gene family of potassium ion transportersTwenty-six KUP genes were identified from the whole genome of A.pumila,and 14,14,16 and 40KUP genes were identified from four cruciferous plants,including Arabidopsis lyrata,Arabidopsis helleri,Eutrema salsugineum and Camelina sativa.Phylogenetic analysis showed that the 123 KUP genes were divided into four subgroups.Collinearity analysis within species revealed that the ApKUP family was mainly underwent the whole genome replication(WGD)/fragment duplication,and purification selection was the main driving force during evolution.RNA-seq data from 15 tissues showed that more than one-third of the gene members were highly expressed in young roots,followed by seed and pedicel tissues.RNA-seq data analysis of five different growth stages showed that many KUP members functioned in plant rapid growth,and their expression levels were significantly up-regulated at pod setting stage(GS4 and GS5),indicating that KUP genes may play important roles in plant growth and development regulation.Gene expression analysis using the RNA-seq data revealed that:(1)The expression of ApKUP gene significantly responded to high salt(250 m M Na Cl),drought(10%PEG6000),low temperature(4?),and high temperature(40?),showing complex change patterns;(2)Under potassium deficiency,except for the up-regulated expression of Ap HAK5,ApKUP1.2 and ApKUP6.1,other ApKUP geneswere down-regulated;(3)50?mol/L Me JA significantly affected the expression of ApKUP in cotyledons and roots,while 1?mol/L ABA significantly affected ApKUP expression in roots;(4)1?mol/L methyl viologen significantly affected the expression of ApKUP in roots and cotyledons.The expression of ApKUP genes responded positively to abiotic stress.However,expression bias between the replication gene pairs suggested the functional conservation and divergence during evolution.To sum up,we established the genetic transformation methods of A.pumila and screened the internal reference genes for qRT-PCR;based on RNA-seq,differentially expressed genes and metabolic pathways involved in growth regulation,such as photosynthesis and circadian rhythm,as well as multiple stress tolerance related genes,such as P5CS and KUP gene family members,were identified in this study.These identified genes were up-regulated in response to stress during growth and development,which endow A.pumila with the adaptive mechanism of rapid flowering and maturation in Xinjiang desert environment.The research system established in this study can provide theoretical reference for the improvement engineering system and crop breeding technology system of breaking through the marginal land.The genetic resources mined in this study can be used in plant resistance genetic engineering breeding,and provide a theoretical basis for accelerating crop breeding strategies,ideas for the research and development of more ephemeral plant resources.This study contributes to the improvement of agricultural biological environment and improvement in Xinjiang in the future.