| Low temperature is one of the common abiotic stresses,which seriously affects the growth and development of plants.In the process of long-term evolution,plants have gradually formed an efficient and complex molecular regulation mechanism in order to resist the external low temperature environment.Environmental stress often leads to the destruction of mitochondrial oxidation defense system,the obstruction of electron transport in respiratory chain and the inhibition of respiratory pathway,thus reducing the respiratory rate.There are few reports on the molecular regulation mechanism of energy metabolism in alpine plants.In this study,callus of Saussurea laniceps was used as experimental material to measure the respiratory rate of Saussurea laniceps callus during low temperature acclimation.Based on the low temperature transcriptome database,he purpose of this paper is to clarify the molecular regulation mechanism of energy metabolism of alpine plants adapting to low temperature.The main results are as follows:1.Low temperature acclimation leads to the change of respiratory rate.The results of Seahorse XF96 energy metabolism analyzer showed that the respiration rate and glycolysis rate of Saussurea laniceps callus decreased significantly in the early stage of low temperature acclimation(4 ℃)(6 days),but the respiration rate and glycolysis increased slightly with the extension of acclimation time to 9 days,but still lower than the control level.Through the addition of inhibitors sodium azide and salicyloxime acid to the callus respiration system,it was found that the proportion of cytochrome C pathway in total respiration of Saussurea involucrata calli cultured at room temperature and acclimated at 4 ℃ for 6 days and 9 days was 86.21%,82.11% and 80.40%,respectively.The proportion of alternative respiratory pathway in total respiration increased from 13.79% to 17.89% and19.60%,indicating that low temperature acclimation significantly increased the proportion of alternative respiratory pathway in Saussurea laniceps callus.2.Identification of genes related to glucose metabolism.Based on the sequencing data of low temperature acclimation transcriptome of Saussurea laniceps callus,47 differential Unigene genes related to glucose metabolism were screened,including glycolysis,tricarboxylic acid cycle and pentose phosphate pathway.For example,low temperature acclimation increased the expression of hexokinase(HK)related to glycolysis pathway,and the expression of phosphofructokinase(PFK)increased in varying degrees after low temperature acclimation.The gene expression of gluconolactone(PGLS)and6-phosphogluconate dehydrogenase(PGD),the key enzymes of phosphate pentose pathway,increased significantly after acclimation at low temperature.3.Screening of differential genes related to mitochondrial respiratory chain and isolation and purification of mitochondria.A total of 32 differential genes related to respiratory chain were screened,among which the expression of AOX1 and AOX2 in(AOX)family,which is the key enzyme of cyanide resistant respiratory pathway,was significantly increased.At the same time,the mitochondria of callus were isolated and purified by discontinuous sucrose density gradient centrifugation,and the purity of mitochondria was verified at the gene level.Using the purified mitochondrial genome RNA as a template,it was detected that the expression of genes regulated by mitochondrial genome was up-regulated after low temperature acclimation.4.Screening and comparison of stable internal reference genes.Based on the sequencing data of low temperature transcriptome of Saussurea laniceps,11 candidate internal reference genes were selected and analyzed by Ref Finder combined with Cq,ge Norm,Norm Finder and Bestkeeper.It was found that the relatively stable genes of ABA(75.7 μ M)were UBC36 and SKIPI,while the stability of PP2AA2 and TUA2 was lower than that of;SA((24 μ M).After treatment,TIF3 HI and PP2AA2 were more stable,TUA2 and UEVID were less stable.After JA(50 μ M)treatment,TIF3H1 and UBL5 were more stable,while TUA2 and UEVID were less stable.At the same time,it was found that TIF3B1 and TUB3 were relatively stable under low temperature(4 ℃)stress,while TIF3 HI and TUB8 were relatively stable,TIF3 HI and ELF5 A were relatively stable,TUA2 and UEVID were relatively stable after salt stress(100m M),UBC36 and UBL5 genes were relatively stable under UV treatment,while SKIPI and TUA2 were relatively stable.So the choice of internal reference genes depends on the types of abiotic stress. |
