Analysis Of Physiological Changes And Gene Difference Expression Of Illicicum Difengpi Under Different Drought Stress Conditions

Illicicum difengpi K.I.B.et K.I.M is an endemic plant of karst limestone mountain,mainly growing on the rocks or semi-naked rock on the top of karst mountain,which can adapt to the extreme conditions of the mountain.In this study,using the seeds and seedlings of I.difengpi as materials,to research the effect of water stress(10% ~ 90% soil relative water content range,a difference of 10% as a gradient)on the seed germination and early seedling growth,and the physiological and ecological adaptability of late seedlings responding to drought stress(70%~80% as control,40%~50% and 10%~20% as stress treatment)were studied.RNA was extracted and sequenced using the Illumina Hi Seq platform for the transcriptome sequencing.Afterwards the sequencing results were analyzed,including the GO functional annotations,KEGG pathway annotations and differentially expressed genes between different treatments.Some genes that may be relevant to the drought-resitant tolerance of the I.difengpi was selected.The main results of this study are as follows:1.The I.difengpi seeds could germinate in given soil moisture contents(40%~90%).However,the germination rate was different in different soil moisture contents.The seed germination rate was the highest in 60% and 70% relative soil moisture content.When the soil moisture content was less than 40%,the seeds cannot germinate.But after the water stress was relieved,the seeds could germinate quickly.2.The early I.difengpi seedlings could grow in the soil water content range from 40% to 100%.The height of the plant,the number of the leaf and root,crown width,and biomass showed a trend of first rise and then decrease with the decrease of soil water content,and of 70% the relative soil moisture content was the optimum moisture condition.Meanwhile,the biomass and total biomass of the aboveground and underground parts were also the most under the same condition.3.When the I.difengpi seedlings were under drought stress,its photosynthetic parameters such as net photosynthetic rate(Pn),stomatal conductance(Gs)transpiration rate(Tr) decreased significantly,intercellular CO2 concentration(Ci)was decreased first and then increased.Chlorophyll a(Chla),Chlorophyll b(Chlb)content decreased significantly.But the Car/Chl(a+b)of sample treated with water stress was significantly higher than that of control.The content of proline and soluble sugar increased significantly with the increase of water stress.The activity of SOD and POD continued to increase significantly when soil water content decreased from 70%~80% to 40%~50%,and finally decreased to 10%~20%.However,MDA content increased significantly only when soil water content was 10%~20%.4.Obtained unigene was compared with NR,String,Swissprot,KEGG,Pfam database,and the genes of each database were 38938,13493,15198,22823 and 19398,respectively.In comparison with NR database,the highest rate of species classification was Vitis vinifera.The COG function classification of Unigene divides all unigene into 26 different functional categories,among which the functional genes of general function and signal transduction mechanism were the highest number.The KEGG annotation of the transcriptome will annotate all Unigene to 379 pathways.5.Analysis of differentially expressed genes showed that there were 124 common differentially expressed genes in all samples,twenty-eight of which were significantly up-regulated and the rest 96 genes significantly reduced.The number of significant differentially expressed genes in 70%~80% vs 10%~20% was the highest,which is 4451,The second was 70%~80% vs 40%~50%,and there were 2489 differentially expressed genes.Moreover,the differentially expression genes between 40%~50% and 10%~20% was the least,and the number was only 753.6.A total of 16218 SSR were obtained by SSR analysis,of which 8644 were the single base repeats.SNP analysis showed that the number of SNP in 9 samples was 88707,93711,68072,61398,77649,66656,79726,84947,75883 in the following order 70%~80%(1-3),40%~50%(1-3),and 10%~20%(1-3)respectively.7.Through analysis of differentially expressed genes between sample,five categories transcription factor genes(79 genes)associated with drought-resistant(b ZIP,DREB,MYB/MYC,NAC,WRKY),2 continuously significantly up-regulated and high expressed level genes of IV chitinase,and six glyceraldehyde 3-phosphoric acid dehydrogenase gene were screened.These genes may be involved in the adjustment process of I.difengpi response to drought stress.