低温胁迫对不同耐寒型甘蔗蔗糖代谢相关酶基因表达及其酶活性的影响

Effect of chilling stress on sucrose metabolism enzymes in leaf of sugarcane varieties with different cold resistance

[目的]研究不同耐寒型甘蔗品种叶片中蔗糖代谢关键酶基因表达及其酶活性在低温胁迫条件下的变化,为培育高产高糖、抗寒性强甘蔗新品种提供参考.[方法]以耐寒型甘蔗品种GT28和冷敏感型品种ROC22为材料,幼苗期进行6℃低温胁迫处理0（对照）、2、5、9 d及胁迫处理5和9d后恢复生长2d,分析两品种甘蔗＋1位叶叶片蔗糖代谢关键酶基因表达及相关酶活性.[结果]与对照相比,低温胁迫利于诱导GT28相关酶基因的表达,而抑制ROC22相关酶基因的表达.6℃低温胁迫2、5、9 d后,GT28叶片SAI、CIN和SPS1基因相对表达量呈先降低后升高的变化趋势,而SPS2和SS表达量呈逐渐升高的趋势,分别以低温胁迫2和9d最高,为对照的8.31、6.99、3.80、5.27和1.65倍;冷敏感型ROC22叶片SAI、CI和SPS2基因相对表达量呈不断下降的变化趋势,以胁迫2d的相对表达量最高,分别为对照的9.79、5.37和4.711倍,而SPS1和SS相对表达量分别呈上升、先上升后下降的变化趋势.低温胁迫5d后恢复生长2d的ROC22叶片SAI、CIN、SPS1和SS及GT28叶片SPS1和SPS2基因相对表达量明显高于胁迫9d后恢复生长2d的处理,而GT28叶片SAI、CI和SS的表现相反.在胁迫5、9d后恢复生长2d,ROC22叶片CIN、SPS2均有所上升,GT28叶片SAI、SPS1和SS相对表达量均有所下降.与对照相比,低温胁迫2～9 d可不同程度提高不同基因型甘蔗品种叶片AI、NI、SPS和SS酶活性,AI和NI酶活性以GT28叶片相对较高,而SPS和SS酶活性以ROC22叶片表现更高.随胁迫时间的延长（2～9d）,GT28叶片的AI、SPS和SS酶活性均呈逐渐上升的变化趋势,以胁迫处理9 d最高,分别为161.62 μmol Glc/gFW·h、7.88和14.28μmol Suc/gFW·h,而NI酶活性以胁迫处理2 d最高（472.49 μmol Glc/gFW·h）.ROC22叶片AI和NI酶活性随胁迫时间的延长而逐渐降低,均以胁迫处理2d的最高,胁迫处理9d的最低;SPS和SS酶活性则表现为先上升后下降的变化趋势,以胁迫处理5d时最高,分别为54.72和81.15 μmol Suc/gFW·h.此外,胁迫处理9 d后恢复生长2 d的GT28和ROC22叶片AI、NI和SS酶活性均高于胁迫5d后恢复生长2d的处理;与胁迫5d处理相比,恢复生长2 d后ROC22叶片AI、NI和SS酶活性均有所降低,而SPS活性有所升高.[结论]低温胁迫条件下,GT28能够提高蔗糖代谢相关酶基因的表达和主要酶活性,利于平衡蔗糖代谢过程,更好适应低温环境.

[Objective]The present study was conducted to investigate the changes in related-gene expression and activities of sucrose metabolism key enzymes in sugarcane leaves under low temperature stress in order to provide the-oretical references for breeding new high-yield, high-sugar sugarcane varieties with strong cold resistance. [ Method ]Sugar- cane varieties GT28 （strong cold resistance type） and ROC22 （cold sensitive type） were used as the tested materials. When sugarcane grew at 6-7 leaf stage, all seedlings were kept in the room of 6~C for 0, 2, 5 and 9 days, and recovering growth for 2 days after stress for 5 and 9 days, respectively. The ＋ 1 leaves of different treatments were sampled to deter- mine the expression of sucrose metabolism-related enzyme genes viz., sucrose phosphate synthase （SPS）, soluble acid inver- tase （SAI）, cell wall-bound invertase （CIN）, sucrose synthetase （SS） and the activities of sucrose metabolism key enzymes viz., AI, neutral invertase （NI）, SPS and SS. [Result]As compared to 0-day treatment, low temperature stress could in- duce the relative expression level of SAI, CIN, SPS1, SPS2 and SS genes in leaf of GT28, but inhibit those of ROC22. After 6 ℃ stress of 2, 5 and 9 days, the relative expression level of SAI, CIN and SPS1 in leaf of GT28 were found to decrease firstly and increase subsequently, while that of SPS2 and SS presented gradually increasing trend. Different genes had the highest expression level at 2 and 9 days stress, which was 8.31, 6.99, 3.80, 5.27 and 1.65 times of the control, respectively. As to ROC22, the expression level of SAI, CIN, SPS2 genes in leaf showed decreasing during stress stage, and their highest level presented at 2 days of stress, which was 9.79, 5.37 and 4.711 times of the control. SPS2 and SS genes in leaf of ROC22 showed change trends of increase, and from increase to decrease, respectively. After recovering growth for 2 days, the relative expressions of SAI, CIN, SPSI and SS in leaf of ROC22 for stress of 5 days were higher than that of treatment for stress of 9 days, as well as SPS1 and SS in leaf of GT28; while SAI, CIN and SS in leaf of GT28 showed on the contrary. When recovering growth for 2 days after stress of 5 and 9 days, CIN and SPS2 in leaf of ROC22 showed increasing expression levels, while SAI, SPS1 and SS in leaf of GT28 showed on the contrary. As com- pared to 0-day treatment, 6 ℃ stress of 2-9 days could increase the activies of sucrose metabolism key enzymes viz., AI, NI, SPS and SS in leaves of GT28 and ROC22 in varying degree. The activities of AI and NI presented higher in leaf of GT28, while SPS and SS showed higher in leaf of ROC22. With the elongation of stress （2-9 days ）, the activities of AI, SPS and SS in leaf of GT28 were found to increase gradually, and reached the highest under stress of 9 days, which were 161.62 μmol Glc/gFW.h, 7.88 and 14.28 μmol Suc/gFW.h, respectively; while NI activity showed the highest un- der stress of 2 days （472.49 Glc/gFW. h）. The activities of AI and NI in leaf of ROC22 was decreased gradually with the elon- gation of stress, and showed the highest under stress of 2 days and the lowest under stress of 9 days. The activities of SPS and SS in leaf of ROC22 showed change trends of increase following by decrease, and reached the highest under stress of 5 days, which were 54.72 and 81.15 μmol Suc/gFW. h, respectively. Furthermore, after recovering growth for 2 days, the AI, NI and SS activities in leaves of GT28 and ROC22 under stress of 9 days were higher than that of treatment under stress of 5 days. As compared to treatment under stress of 5 days, the activities of AI, NI and SS in leaf of ROC22 showed decrease after recovering growth for 2 days, while that of SPS was found to be increased. [Conclusion ]Under low temperature stress, GT28 could increase expression level of sucrose metabolism-related gene and key enzymes＇ activities, balance the sucrose metabolism process to well adapt low temperature environment.