| Kareliniacaspia is a perennial herb of the genus Karelinia in the compositae family.It is fond of growing in large groups of halophyte grass in desert areas with high temperature.It can be used as forage grass and has strong resistance to salt,drought and high temperature.Through transcriptome sequencing and analysis of Kareliniacaspia leaves treated at room temperature and high temperature,this study explored the genes and biological pathways related to high temperature tolerance of Kareliniacaspia,in order to reveal the molecular mechanism of Kareliniacaspia in response to high temperature stress from the level of gene transcription,and to provide genetic resources and reference for molecular breeding of high temperature tolerance.At the same time,the contents of Ca2+,Mg2+,Na+and K+cations in the roots,stems and leaves of Kareliniacaspia in different growth stages of desert space were determined,so as to reveal the relationship between the dynamic changes of Ca2+,Mg2+,Na+and K+and the high temperature tolerance of Kareliniacaspia.In addition,through to the Kareliniacaspia leaves in 45℃ under the condition of high temperature 0 h,2 h,4 h,8 h,12 h five,five handling leaves extract DNA,RNA and protein electrophoresis analysis,and to handle the five leaf cell protoplast comet electrophoresis test,to explore the biological characteristics of Kareliniacaspia resistance to high temperature and the use of provide a reference basis.In addition,the full-length CDs of Kc_CDPK32,Kc_CIPK9 and Kc_CIPK5 genes were obtained by cloning,and the overexpression vectors were constructed by using bp-lr reaction.The main findings are as follows.1.After treatment at 45℃ for 12 h,the DNA of Kareliniacaspia leaf organs was significantly degraded,and RNA and protein were significantly degraded after treatment for 8 h.By comet electrophoresis,it was found that the Kareliniacaspia leaf cells were deformed and obviously damaged after 8 h treatment.The results showed that the critical time of Kareliniacaspia resistance to 45℃ was 8 h,and it had a strong adaptability,which could be used as an important material of plant resistance to high temperature and an important germplasm resource for ecological restoration and reconstruction in high temperature desert areas.2.(1)By comparing with normal temperature-high temperature transcriptome sequencing,received 68244 Unigene,75 766 075 nt,1110 nt,the average length N50 reached 1756 nt,among them there were 32777 differentially expressed genes(differentially expressed multiples>2),will receive the differentially expressed genes in NR,NT,Swiss-Prot,KEGG,COG,GO(E-value<1.0 e-5)compare the database,such as found that there were 13 593 differentially expressed genes get comments,5358 of which genes are expressed to raise,The expression of 8235 genes was down-regulated.(2)The analysis of these differentially expressed genes,discovery carotenoid biosynthesis,zeatin biosynthesis,plant hormone signal transduction,flavones and flavonols biosynthesis,alpha linolenic acid metabolism,GSH metabolism and biosynthesis of flavonoids,unsaturated fatty acid biosynthesis,the sheath of sugar lipid biosynthesis-ganglio series,sheath sugar lipid biosynthesis-receiving globo series,sphingolipid metabolism,ribosome,endoplasmic reticulum protein processing path,cutin,suberin and wax biosynthesis and metabolic pathways of differentially expressed genes closely associated with Kareliniacaspia high temperature resistant ability.(3)Based on the analysis of the obtained differentially expressed genes,it was found that there were 74 differentially expressed genes related to Ca2+transport,among which 24 were up-regulated and 50 were down-regulated.3.Through the experiment,it was found that the K+/Na+ ratio in the root and stem organs of Kareliniacaspia had a large change range,ranging from 0.45 to 1.64,while the ratio in the leaf organs had a small change range,ranging from 0.12 to 0.43.It is speculated that the large variation of K+/Na+ratio in Kareliniacaspia may be related to its salt tolerance.The ratio of K+/Na+ in stem and leaf decreased significantly during flowering period(high temperature),suggesting that the decrease of K+/Na+ ratio was related to high temperature.The ratio of(Ca2++Mg2+)/(Na++K+)in each organ of Kareliniacaspia was the highest in the flowering period(high temperature),during which the content of Na+ and K+ was relatively low,while the content of Ca2+and Mg2+was relatively high.These results suggest that Kareliniacaspia can improve its high temperature tolerance by selective absorption of Ca2+and Mg2+plasma at high temperature.4.(1)The full length of gene CDs of Kc_CDPK32,Kc_CIPK5 and Kc_CIPK9 were obtained by experiment and sequencing.The total length of Kc_CDPK32 gene was 1587bp,encoding 528 amino acids.Kc_CIPK5 gene is 1356bp long and encodes 451 amino acids.Kc_CIPK9 gene is 1320bp long and encodes 439 amino acids.(2)Three genes were constructed into PK2GW7 plant expression vectors by using BP-LR technology,and PK2GW7-Kc_CDPK32,PK2GW7-Kc_CIPK97 and PK2GW7-Kc_CIPK5 gene expression vectors were stored in agrobacterium GV3101 and agrobacterium EHA105. |
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