Molecular Basis Of Resistance To ScMV And Environmental Risk Assessment Of ScMV-CP Gene Transgenic Sugarcane

Sugarcane mosaic disease is one of major disease to sugarcane and influences the growth of sugarcane seriously. Breeding resistant cultivars by genetic engineering is an assistant method in high-effective breeding of sugarcane. ScMV-CP gene was transformed to high-susceptible sugarcane cv. Badila by particle bombardment in the Key Lab of Sugarcane Eco-Physiology& Genetic Improvement, Ministry of Agriculture in 2003, and 13 ScMV-CP gene transgenic sugarcane were gained. Using these transgenic sugarcanes as material, the resistant stability and agronomic performance of transgenic sugarcane were studied after environmental release; and photosynthetic basic of yield increase was studied through the difference of photosynthetic characteristics between transgenic and non-transgenic sugarcane; After resistance identifying repeatedly, the high-resistant transgenic line was chosen to study the physiological and biochemical basis and molecular basic of resistance; and transgenic sugarcane environmental risk was assessed as well. The results showed as followed:Resistance to ScMV was improved significantly in CP transgenic sugarcanes, the incidence of ScMD(sugarcane mosaic disease) ranged from 0 to 34% after inoculation and natural infection, and kept stable in different generation of progenies. But the incidence of the non-transgenic sugarcane amounted to 92-100%. 5 transgenic lines were chosen for environmental release according to the agronomic performance and resistance of transgenic sugarcane, the results indicated that the incidence of ScMD ranged from 0 to 20%, stalk length increased by23-32%, effective stalk increased by 45-55%, cane yields of per hectare increased by 62-80.6%, and the sugar content(absolute value) increased by 1.15-2.15% in transgenic sugarcane compared with the counterpart in non-transgenic sugarcane. The transgenic line B48 was the best line in 13 transgenic sugarcane lines, and had nice agronomic traits and no disease development after inoculation or natural infection in four years.The cell ultrastructure, chloroplast proteome and photosynthetic traits in leaves of transgenic line B48 and the counterpart of non-transgenic line were studied. The results showed that the cell ultrastructure of non-transgenic sugarcane leaves displayed the typical cytopathological character infected by ScMV, and the cytoplasm was filled with ScMV particles, but none of these profiles were found in cell ultrstructure of transgenic sugarcane. These confirmed that the sugarcane mosaic disease was caused by ScMV and showed the disease resistance of the transgenic line on cellular level in addition. The chloroplasts were destroyed and the grana lamella disappeared in non-transgenic sugarcane leaves after infected by ScMV. The chloroplast proteome was studied by 2-DE. it was found that the chloroplast photosystem I protein and Oxygen evolution complex(OEC) of photosystem II(PS II) in leaves of non-transgenic sugarcane were down-regulated. These suggested that the PSI and PS II of chloroplasts in leaves of non-transgenic were damaged after virus infection, and resulted in the decrease of chloroplasts content and net photosynthetic ratc(Pn). Moreover, the infection lowered the activity of phosphoenolpyruvate carboxylase (PEPC). All of these changes in leaves of non-transgenic sugarcane decreased the CO₂ assimilation capacity and inhibited the plant growth, and finally caused the yield loss. While transgenic sugarcane had integrated cell structure and chloroplasts structure and accompanied with the high content of chloroplasts and activity of PEPC in leaves. So the transgenic line kept better capacity of photosynthesis. It can be regarded as the photosynthetic basis of the yield increase in transgenic sugarcanes.The content of endogenous free SA and conjugated SA increased both in transgenic and non-transgenic sugarcane after inoculation. And SA had a significantly negative correlation with CAT activity, increasing of SA inhibited the CAT activity and caused accumulation of H₂O₂. These were the common defense response to ScMV in transgenic and non-transgenic sugarcane. But the increase of free SA in transgenic sugarcane was higher than non-transgenic sugarcane, and ahead of the latter. At the same time SOD, POD activity increased and CAT activity lower remarkably in transgenic line. Changes of these enzyme activities leaded to accumulation of H₂O₂ and enhanced the cell wall cross—linking to limited the virus to infect further. But in non-transgenic sugarcane, the SOD,POD activity increased slowly, and decreased rapidly, and CAT decreased slowly. These changes of protective enzyme activities influenced the accumulation of H₂O₂ and cause the plant could not establish signal transduction pathways of resistance effectively.The studies of metabolism of phenylalanine showed the PAL, PPO activities enhanced both in transgenic sugarcane and non-transgenic sugarcane after inoculation with ScMV. PAL activity in transgenic line increased rapidly and kept stable after inoculation. But PAL activity in non-transgenic increased later than transgenic line and decreased promptly. And PPO activity in non-transgenic sugarcane was later and lower increase than transgenic sugarcane. The content of flavonoids in leaves accumulated fast, last a long time and kept a higher concentration in transgenic sugarcane after inoculation; But the content of flavonoids decrease and can not keep stable level in non-transgenic line. Metabolism of phenylalanine in transgenic sugarcane differed from non-transgenic sugarcane are cause by the different resistance to the ScMV.Different genes expression in transgenic and non-transgenic sugarcane was analyzed after inoculation used E. arundinaceum(sugarcane related plant) cDNA microarray under drought stress developed by our lab. The result showed there were 43 up-regulated clones in transgenic sugarcane. These clones took part in cell structure protection, signal transduction, protein synthesis, energy metabolism, respiration and so on. For example, cell wall protein GP2(HRGPGP2) and protein binding / structural constituent of cell wall played a role in cell structure protection, and zinc finger protein, MYB protein transcription factor took part in resistant signal transduction, and acetyl CoA carboxylase participated in secondary metabolism to produce flavonoids and other secondary metabolites. These results suggested that the resistance to ScMV in transgenic sugarcane due to resistant genes were activated rather than transferred CP gene into sugarcane. Candidate MYB gene fragment was cloned base on result of cDNA mircroarray hybridization. Bioimformatics analysis of the fragment suggested the sugarcane MYB gene belonged to the typical R2R3 MYB family genes. And result of real-time quantitative PCR analysis indicated the sugarcane MYB gene did not show any organ-specific, but induced by exogenous SA and H₂O₂ strongly. These suggested this MYB transcription factor of sugarcane may play a role in the resistance.Study on proteome in leaves of transgenic line B48 and non-transgenic line showed that images of 2-DE were very different from each other after inoculation. 10 proteins were up-regulated and 1 was induced specifically in leaves of transgenic sugarcane, but 3 were up-regulated and 1 was induced specifically in leaves of non-transgenic sugarcane. 7 different proteins were identified by MS/MS using 4700 Proteomics Analyzer (Applied Biosystems, USA). The up-regulated proteins in leaves of transgenic sugarcane were IkappaB kinase, cytosolic ascorbate peroxidase (APX) and Mn-superoxide dismutase (Mn-SOD), and unknown protein was specifically induced. The up-regulated proteins in leaves of non-transgenic sugarcane were unknown protein and rieske Fe-S precursor protein, and drought-inducible protein was specifically induced. These suggested that the different protein expression due to the different resistance of transgenic sugarcane and non-transgenic sugarcane.Environmental risk of ScMV-CP transgenic sugarcane was primarily assessed thought non-target organism investigation in the field, CP gene horizontal transfer to the soil microbe and effects on micro-ecology of rhizosphere soi in different growth stage of sugarcane and under different cultural patterns. Effects on micro-ecology of rhizosphere soil were studied using BIOLOG Ecoplate and plate cultivation of microorganism associated with related soil enzyme activities analyses. The results showed that there was no difference in pest harming between transgenic sugarcane and non-transgenic sugarcane, and no ScMV-CP gene was detected in soil total DNA; The total number of colony forming units(CFUs) of culturable bacteria and fungi were increased in transgenic sugarcane rhizosphere soil, but the total number of CFUs of culturable actinomycetes was not effected. Changes of CFUs of microbe resistant to kanamycin(Km) in medium indicated that number of soil microbe resistant to Km in soil wasn't increased because of NPT II in transgenic sugarcane; BIOLOG experiment showed the metabolism activity of soil bacteria was increased lightly, and the soil bacteria diversity indexes such as Simpson index(D'),Shannon-Wiener index(H'), evenness, Mclntosh index(DMc) were not different between transgenic sugarcane and non-transgenic sugarcane after inoculating 72h in BIOLOG Ecoplate. Urease activities in the rhizosphere soil of transgenic sugarcane were significantly higher than that of non-transgenic plant, but sucrase activity and phosphatase activities were not influenced. These primary results indicated that transgenic sugarcane had some effects on soil bacteria and fungi, but on effect on actinomycetes; transgenic sugarcane had no disadvantage influence on soil fertility at the present time. These suggested changes of micro-ecology of rhizosphere soil were caused unlikely by exogenous gene expression in transgenic sugarcane, maybe was the non-prediction effect after environmental release of transgenic sugarcane.