| Cassava(Manihot esculenta Crantz)is one of the most important tropical root crops,and its yield is closely related to the size,number and starch content of root.At the same time,its yield is affected by biotic stress and abiotic stress.Sugar Transport Proteins(STP)are proteins which are responsible for the transport of monosaccharide and involved in their tissue development and stress response in plants.It is important to analyze the physiological and molecular mechanism of cassava tuber development and adaptation to low temperature,and to dig out genes involved in root development and response to cold damage.It has important theoretical significance and application prospect for genetic improvement of cassava and breeding of high-yield and cold resistant varieties.In this study,cassava(KU50)was used as the experimental material to analyze the development process of cassava root tuber and physiological changes under low temperature stress.On this basis,the MeSTP17 and MeSTP19 were screen out by real-time PCR technique,which were high expression at the root formation period and were up regulated by low temperature.The total length of cDNA was cloned.The transport characteristics were analyzed by subcellular localization and yeast mutant functional recovery.Finally,the functions of these two genes in plant root development and low temperature stress are studying through the transgenic method.The main findings are as follows:(1)By observing and analyzing the root system of cassava from 10 days to 203 days after planting,it was found that most of the fibrous root were degenerated and disappeared,and a little part gradually developed to root tubers.The total number of roots increased first and decreased gradually after 45 days after planting.The total length of roots first increased,and decreased gradually on the 45 day after planting.The diameter of roots increased sharply on the 45 day after planting.The total number of root tubers began to gradually increase in the 45 day after planting and increased to 10 to 11 remained relatively stable.In the root of 45 days after planting,the primary fibrous root,the secondary fibrous root,the root tuber and different growth areas of the same root in the local expansion including the bulk,the thickening part and the non thickening part.It shows that the root development of 45 days after planting is in the early stage of root formation and the root morphology is changing.(2)It was analyzed that the activity of acid invertase and sucrose synthase and the change of sugar content in the root development of 45 days after planting.The activity of cell wall acid invertase:primary fibrous root>secondary fibrous root>root tuber,primary zone>secondary zone>enlarged zone;intracellular soluble acid invertase activity:primary fibrous root>secondary fibrous root>root tuber,primary zone<enlarged zone<secondary zone;sucrose synthase activity:primary fibrous root<secondary fibrous root<root tuber,primary zone<enlarged zone<secondary zone;fructose,glucose and sucrose content:primary fibrous root<secondary fibrous root<root tuber,primary zone<secondary zone<enlarged zone.The above results suggest that the way of sugar unloading occurred and more carbohydrates need to be involved during the formation of tuber root.(3)The expression pattern of cassava MeSTPs gene family members were analyzed in roots of the 45 day after planting.The relative expression of MeSTP17 and MeSTP19 in three morphological roots was significantly higher than that in other members.By analyzing the expression patterns of the STP family members in the different growth regions of the same root in local expansion,the relative expression levels of MeSTP17 and MeSTP19 in different growth areas were significantly higher than those of other members.These results imply that MeSTP17 and MeSTP19 have certain root tissue specific expression and participate in the early development of cassava roots.(4)The morphological and physiological changes of cassava plants at 0 h,6 h,9 h and 30 h were observed and analyzed under low temperature stress at 7?.It was found that with the prolongation of the low temperature stress time,the tapioca plants were full from the tip buds,and the mature leaves gradually appeared to soften the mature leaves,lose the strength of the petioles down,and the tender stems and leaves wilting.The relative conductivity of the leaves of the cassava leaves increased gradually,and the concentration of MDA decreased slightly after 6 h,but at 9 h.At 30 h,the MDA concentration increased by more than two times.The content of chlorophyll in cassava leaves gradually decreased,but the content of soluble sugar increased gradually,and the content of fructose increased rapidly after 6 h;the content of glucosamine decreased first,increased at 30 h,and the content of sucrose increased gradually.The above results indicated that low temperature stress destroyed the plasma membrane of cassava leaves,hindered photosynthesis and blocked the synthesis of carbohydrates,resulting in the change of sugar metabolism.(5)By analyzing the expression patterns of cassava STP gene family members at 0 h,6 h,9 h and 30 h under low temperature stress at 7? it was found that the expression of the gene was changed in varying degrees.The expression of MeSTP17 and MeSTP19 has a large amplitude.The expression of MeSTP17 is up and then down and the expression in 9 h is up to 78.8.The expression of MeSTP19 is up and the expression of 30 h is up to 21.7.There was no significant change in the expression of other genes.It was preliminarily determined that MeSTP17 and MeSTP19 were involved in the response of cassava to low temperature stress.(6)Using Saccharomyces cerevisiae transport protein mutant,we studied the sugar absorption function of MeSTP17 and MeSTP19.The results showed that MeSTP17 and MeSTP19,except xylose,had strong absorbability to glucose,fructose,mannose and galactose,and were sensitive to the performance of anticoupling agent CCCP.The results show that MeSTP17 and MeSTP19 have broad spectrum absorption characteristics of monosaccharide,and their transport depends on the cross-membrane proton power potential established by H+-ATP.In addition,it was confirmed that the location ofMeSTP17 and MeSTP19 is in cell membrane.(7)Constructing plant over-expression vectors of MeSTP17 and MeSTP19 is transfered to Arabidopsis thaliana,which have been screened to the T2 generation,obtained 19 and 21 lines of transformation seedlings of Arabidopsis thaliana respectively.Above all,the monosaccharide transporter gene of cassava MeSTP17 and MeSTP19,was highly expressed in the early stage of cassava root,and also responded to low temperature stress.The monosaccharide substrates have broad spectrum transport characteristics and are located in cell membrane.It is speculated that MeSTP17 and MeSTP19 are mainly involved in the early development of cassava tuberous roots,affecting root morphogenesis and responding to low temperature stress. |
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