| Agave SPP is a tropical fiber crop widely cultivated in the world,but the low temperature in winter is the key factor limiting the production of sisal in China.In this paper,sisal seedling in different areas was taken as the research object,and the changes of seven physiological and biochemical indexes related to cold resistance of sisal seedling were measured under low temperature stress.The relationship between seven physiological indexes and cold resistance of sisal seedling was analyzed,and the expression of cold-resistance related genes was studied by molecular biology techniques.The main experimental results are as follows:1.Effects of low temperature stress on physiological and biochemical indexes of Sisal hempIt was found that the semi-lethal temperature of sisal seedling was 3℃,and the damage of sisal seedling would be irreversible if the temperature was lower than 3℃.The content of free Pro in sisal increased gradually with the decrease of temperature,but there was no significant difference between 6℃ and 9℃.The temperature at 3℃was significantly higher than that of the control.It reached the first peak value at 18 h,and then showed a slow upward trend from 18 h to 24 h.After low temperature treatment,sisal seedling 9℃,6℃ and 3℃ showed a downward trend,indicating a response of chlorophyll to low temperature.At 3℃,there is a significant decrease.When the temperature is high,the trend of decline is small and relatively cold resistant.The relative cold resistance of continuous decline is weak.Under low temperature stress,the content of malondialdehyde(MDA)in leaves of SISal h.11648 seedlings increased as compared with the control(24℃).During cold stress,the content of soluble sugar in sisal H.11648 seedlings increased with time.The soluble protein content in sisal leaves increased with the extension of time during cold stress.Compared with the control,the SOD activity of sisal hemp seedlings under mild low temperature stress changed with the change of temperature and time,first had an upward trend,then decreased.Under low temperature stress,POD activity of sisal seedling increased gradually.The activity increased rapidly between 3℃ and0℃,and the activity decreased at 9℃ and 6 ℃,with no significant difference.After low temperature treatment,CAT in the leaves of sisal showed a significant decrease,and there were significant changes in sisal at each 24 hr temperature stage,with a decrease of more than 50%,reaching a very significant level.However,there was a gentle increase at 12 and 24 hr stage.2.Effects of agents on sisal hemp response to low temperature stressThe determination of chlorophyll content in sisal seedling showed that amino-oligosaccharides were slightly higher than other treatments.Spraying amino-oligosaccharide for different treatment duration had significant effect on growth and physiology of sisal seedling.Application of amino-oligosaccharide was beneficial to increase physiological activity,POD enzyme,SOD enzyme and CAT enzyme activities in sisal sisal leaves at late growth stage.It can prevent early senescence of leaves and provide sufficient photosynthetic material for sisal hemp development.Spraying the whole growth period can improve the cold resistance of sisal hemp.3.CBF gene identification and expression analysis of sisal hemp in response to low temperature stressFive CBF gene family transcription factors were identified from sisal sisal genome by bioinformatics methods,and their physical and chemical properties,phylogenesis,functional elements and gene transcription were analyzed.The results showed that the length of the five CBF proteins ranged from 415 to 1127 AA.Phylogenetic analysis showed that the five CBF genes could be divided into two subgroups,in which group A contained four genes and group B contained only one gene.The expression analysis showed that all the five CBF genes had different degrees of response to cold stress.This study summarized the basic characteristics of CBF gene family in Sisal,and accumulated basic data for further study and application of CBF transcription factors.4.Expression analysis of sisal photosystem genes responding to low temperature stress(1)The expression of photosystem genes was analyzed,and 6.0G of original data were obtained by sequencing.The assembled chloroplast genome length was157268 bp,and GC content was 37.85%.The length of the LSC is 85894 bp,the length of the two IR regions is 26573,and the length of the SSC is 18228 bp.A total of 86protein-coding genes,38 t RNA and 8 r RNA sequences were annotated.(2)The contents of soluble sugar,soluble protein and proline all decreased with the decrease of stress temperature,and the change of proline content was the most obvious at 3℃,which decreased by 95% compared with the control.(3)The activities of antioxidant enzymes in sisal leaves decreased significantly at low temperature compared with the control,and the activities of SOD and POD decreased by 11.6% and 97.1% at 6℃(4)Chlorophyll content basically showed a decreasing trend with the decrease of temperature.Under low temperature stress,chlorophyll content in sisal leaves decreased,photosynthetic electron transfer rate decreased,and heat dissipation increased,indicating that the photosystem was damaged.The expression levels of light-trapping chlorophyll A-B binding proteins(psd A and psd B)genes were down-regulated,and the number of pigment-protein complex was decreased,which reduced the damage of low temperature to the photosystem to a certain extent.With the decrease of temperature,osmotic regulation substances(SS,SP and Pro)were mainly used for respiration,and the activity of antioxidant enzymes(SOD and POD)gradually decreased,and the metabolic level of cells decreased,and the degree of damage increased.The expression of fatty acid desaturase gene was up-regulated to maintain the stability of biofilm system by increasing the unsaturation of fatty acid.5.Comparative study on cold resistance of sisal in different regionsSisal seedlings from 11 regions were sampled and treated at 5℃for 24 h between cold-resistant lethal temperature and semi-lethal temperature.Physiological and biochemical indexes of sisal seedlings were measured.The chlorophyll index,membrane permeability index,malondialdehyde(MDA)with the most significant change,and the enzyme activity index,superoxide dismutase(SOD)with the most significant change were selected.The data from 11 regions were sorted out and compared,and the cold resistance of sisal seedling in different regions was preliminarily judged. |
