| Ammopiptanthus nanus is one of the two species of Ammopiptanthus in Leguminosae.It is only distributed in Wuqia County,Xinjiang,and is the only evergreen broad-leaved shrub in the desert.It is a national first-class protected plant,which has important scientific value for the study of the occurrence and development of desert flora in southwest Xinjiang.It has long been adapted to the harsh natural environment such as drought,high temperature and low temperature,which makes it a better material for mining resistance genes.In this study,the effects of drought stress on seed vigor and physiological and biochemical indexes of Ammopiptanthus nanus were studied,and the seedlings of Ammopiptanthus nanus were subjected to drought stress at different times.The transcriptome sequencing and differential expression gene analysis were carried out to further clarify the response mechanism of seedlings to drought stress.Finally,the stress-resistant extended protein genes were screened out,two genes,AnEXPA1 and ANEXPA2,which were expressed differently The genes related to stress AnEXPA1 and AnEXPA2 were choosed and transferred to Arabidopsis plants to analyze the tolerance of the transgenic plants to cold and drought stress.The main findings are as follows:1.The method of silica gel dehydration was used to dry the seeds of Ammopiptanthus nanus.The seeds with different water content(9.0%,5.4%,4%,4.3%,3.3%,2.6%)were obtained.The germination rate,germination index and vigor index of the seeds with different water content were measured.There was no significant difference in seed vigor,SOD,POD,CAT activity and MDA content when the water content was higher than 4.3%.As the water content was lower than 4.3%,SOD,POD and CAT activity were decreased significantly,and MDA content was increased.The contents of soluble sugar and soluble protein in seeds did not change much in seeds with different water contents.The results showed that the seed vigor and antioxidant enzyme activity would decrease sharply when the water content of Ammopiptanthus nanus seed was lower than a certain threshold,but the content of soluble sugar and soluble protein would not change much.2.Transcriptome sequencing was performed on the Ammopiptanthus nanus seeds at different time(0 h,1 h,6 h,12 h)under drought stress.After quality control screening of the original sequencing data,the number of bases in each treatment was greater than 61.28 Gb.After assembly,Contig 186078959bp,Transcript 175397,492bp,Unigene 57118941bp were obtained.According to the NCBI data,Unigene annotated soybeans(Glycine max)to 10.77%,and peanuts(Arachis ipaensis)to 3.39%.A total of 40607 genes were annotated in the Ammopiptanthus nanus by the GO database.These genes are divided into 46 categories.Unigene was compared and classified by the egg NOG database.A total of 64946 genes were classified into 25 categories.After comparison with the KEGG database,a total of 6173 Unigenes were compared,which were divided into 38 categories.3.The expression difference of Unigene was analyzed by DESeq(version 1.18,0),and the difference of Unigene was screened according to the multiple change of expression amount(| fold change|>2)and the significant change of expression amount(p-value<0.05).The number of differentially expressed genes was the most in D0(control)and D1(drought stress for 1 h),with a total of 6699,of which 5547 were up-regulated and 1152 downregulated.The least differentially expressed genes were D2(drought stress for 6 h)and D3(drought stress for 12 h).The differentially expressed genes were 1552,accounting for 2.28%of Unigene,of which 249 were up-regulated and 1303 were down-regulated.The differentially expressed genes were compared in the GO database.A total of 3,561 genes were annotated compared to D0(control)and D1(drought stress for 1 h).A total of 3,953 genes were compared to D0(control)and D2(drought stress for 6 h).It was noted that 3599 genes were annotated compared to D0(control)and D3(drought stress 12 h).The first 30 GOs that were significantly enriched were analyzed,and the differentially expressed genes were mainly enriched in three aspects:biological processes,cell components,and molecular functions.A total of 832 genes were enriched in 191 pathways in the KEGG pathway compared between D0(control)and D1(drought stress for 1 h).A total of 1361 genes were enriched in 214 pathways between D0(control)and D2(drought stress for 6 h)treatments,and a total of 1102 genes were enriched in 212 pathways in D0(control)and D3(drought stress for 12 h)treatments.The isoflavonoid biosynthetic pathways,starch and sucrose synthesis pathways,and hormone signal transduction pathways of differentially expressed genes were analyzed after Ammopiptanthus nanus roots under drought stress.The genes of isoflavone biosynthesis pathway,starch and sucrose pathway and ethylene pathway were up-regulated,which indicated that these substances played an important role in drought resistance.Nine expansin genes related to stress tolerance were screened out,and two expansin genes showed significant changes in expression level,which deserved further study.4.Based on the RNA-seq analysis,nine expansin genes related to stress tolerance were screened out,and two expansin genes showed significant changes in expression level,which were chose for further study.Two extended protein genes screened from RNA-Seq data of Ammopiptanthus nanus root under drought stress,named AnEXPA1 and AnEXPA2 respectively.Both genes had a 747-bp CDS sequence and encoded a polypeptide of 248 aminoacids,with predicted molecular weights of approximately 26.53 and 26.69 kD.They respond to different abiotic stresses and hormone signals and may play an important role in coping with abiotic stresses.Glucuronidase(GUS)staining was analyzed showed that AnEXPAl promoter activity was induced only under low temperature stress,while AnEXPA2 promoter activity sensitive to hormone induction.Moreover,transgenic Arabidopsis expressing AnEXPA1 and AnEXPA2 genes drivern by 35S promoter dispayed stronger resistence to cold and drought stree compared with wild type Arabidopsis,suggesting that AnEXPA1 and AnEXPA2 play an important role in the response to abiotic stress.This study provides new idea for biological control and molecular breeding of desertification in Leguminosae. |
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