公主岭霉素诱导对育苗期水稻耐冷性的影响

Induction of Cold Tolerance in Rice at the Breeding Stage by Gongzhulingmycin

【目的】探讨在低温条件下公主岭霉素对水稻幼苗生长及耐冷相关基因表达、抗逆防御酶活性的影响,明确施用公主岭霉素后育苗期水稻耐冷性的变化。【方法】以‘吉粳88’为试验材料,经公主岭霉素水浸提液浸种引发后播种,调查不同温度下稻种萌发和幼苗生长状况并计算稻种发芽临界温度。将农抗“769”固体发酵物干燥粉碎后添加到水稻育苗基质中,以‘吉粳88’为试验材料,在立针期模拟倒春寒生境对水稻幼苗施加冷胁迫处理,在处理后逐渐缓慢升温并于1—8 d内连续采样,升温至28℃后每隔7 d采样一次;以‘吉宏6号’为试验材料,大棚育苗待水稻幼苗长至一叶一心期采样;采用实时荧光定量PCR(RT-qPCR)技术,分析模拟生境及大棚育苗环境中幼苗叶片耐冷相关基因OsNAC6、OsSADMC、OsETR4、OsZFP151的表达状况,并检测移栽前大棚秧苗叶片中防御酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)活性的变化。【结果】公主岭霉素能够提高稻种发芽率和发芽指数,缩短发芽时间,其对幼苗生长的促进作用随温度的降低而逐渐明显,G-500X表现最好,使稻种萌发的临界温度降低了4.09%,并以该浓度为基础进行后续的试验研究。光照培养箱低温模拟倒春寒气候条件,低温胁迫下公主岭霉素可调控OsNAC6、OsSADMC、OsETR4表达量的提升,并通过加快响应速度,实现快速反应以应对低温胁迫,对照处理主要通过调控OsZFP151的响应速度和表达量提升应对低温胁迫,但公主岭霉素处理下的水稻幼苗该基因表达量亦得到提升。水稻幼苗在立针期受到低温胁迫后,公主岭霉素处理的幼苗在低温胁迫后OsNAC6、OsSADMC、OsETR4表达量峰值出现时间较对照早1—2 d,且分别比对照提高38.57%、74.66%、130.61%;OsZFP151最高表达量的时间较对照迟2 d,但最高表达量比对照提高了34.91%。大棚育苗,固体发酵物干粉在育苗基质中的最适添加量为8 g·m^-2,添加后一叶一心期的水稻幼苗叶片中OsNAC6、OsSADMC、OsETR4、OsZFP151的表达量均高于对照,其中OsNAC6、OsSADMC、OsETR4表达量显著提升,OsNAC6在添加量为8 g·m^-2时表达量最高,为261.20;当固体发酵物干粉添加量为5 g·m^-2时,OsSADMC表达量最高,比对照提高了126.30%;当固体发酵物干粉添加量为8 g·m^-2时,OsETR4表达量最高,比对照提高了359.81%。移栽前处于四叶一心期的幼苗,育苗基质中添加公主岭霉素诱导幼苗叶片中SOD、POD、PPO、PAL酶活性均高于对照,其中起主要作用的酶为SOD、PPO,活性分别比对照提高57.18%、28.53%。【结论】公主岭霉素在水稻育苗期施用可降低稻种的发芽临界温度,促进幼苗生长,显著提升幼苗的秧苗素质;公主岭霉素通过提升耐冷相关基因的表达量、加快低温胁迫的响应速度及提高幼苗体内防御酶的活性,促进水稻幼苗耐冷性的增强。

【Objective】The objective of this study is to investigate the effects of gongzhulingmycin on rice seedling growth,cold tolerance genes expression and defense enzyme activities based on chilling stress exposure,and to elucidate the change in cold tolerance induced by gongzhulingmycin at the breeding stage of rice.【Method】Rice variety‘Jijing88’was used as the experimental material,the seed germination and seedling growth were investigated under different temperatures and the critical temperature of seed germination was calculated after seed priming by gongzhulingmycin.The solid fermentation product of gongzhulingmycin was powdered and added into the rice seedling substrate before sowing.Taking‘Jijing88’as the experimental material,the rice seedlings were subjected to cold stress treatment in simulated cold environment in late spring at the needle appearance stage of rice.Gradually warmed up and sampled continuously within 1-8 d after treatment and sampled every 7 days when plant temperature raised to 28℃.Taking‘Jihong 6’as the experimental material,the rice seedlings were raised in the greenhouse and sampled when the seedlings grew to one leaf at a time.The expression of 4 cold tolerance genes(OsNAC6,OsSADMC,OsETR4 and OsZFP151)under the simulated cold environment in late spring and greenhouse seedling environment was analyzed by real-time quantitative PCR(RT-qPCR).The changes of defense enzyme activities such as superoxide dismutase(SOD),catalase(CAT),phenylalanine ammonia-lyase(PAL),polyphenol oxidase(PPO)in greenhouse seedlings before transplanting were detected.【Result】The gongzhulingmycin could improve seed germination rate and germination index,shorten mean germination time,and its promoting effect on seedling growth was gradually obvious with the decrease of temperature.G-500x showed the best performance,which reduced the critical temperature of rice seed germination by 4.09%,and the subsequent experimental study was carried out based on this concentration.At the condition of low temperature simulation of the late spring cold,the application of gongzhulingmycin significantly increased the expression of OsNAC6,OsSADMC,OsETR4,and rapid response could be achieved to cope with low temperature stress by accelerating the response speed.The control mainly regulated the response speed and expression of OsZFP151 to cope with low temperature stress,but the expression of OsZFP151 was also increased after gongzhulingmycin application compared to the control.After the rice seedlings were subjected to low temperature stress at the needle appearance stage of rice,the expression peaks of OsNAC6,OsSADMC and OsETR4 in the seedlings treated with gongzhulingmycin appeared 1-2 d earlier than that in the control,and were increased by 38.57%,74.66%and 130.61%,respectively,compared with the control.The maximum expression of OsZFP151 was 2 d later than that of the control,but the maximum expression was 34.91%higher than that of the control.The optimum added weight of the solid fermentation productive powder of gongzhulingmycin was 8 g·m^-2 in rice seedling substrate in the greenhouse seedling raising.When the seedlings grew to one leaf at a time after the addition of gongzhulingmycin,the expression levels of OsNAC6,OsSADMC,OsETR4 and OsZFP151 in the leaves were higher than those in the control group.The expression of OsNAC6,OsSADMC and OsETR4 was significantly increased.When the added weight was 8 g·m^-2,the expression level of OsNAC6 was the highest(261.20).When the added weight was 5 g·m^-2,the expression level of OsSADMC was the highest,which was increased by 126.30%than that of the control.When the added weight was 8 g·m^-2,the expression level of OsETR4 was the highest,which was increased by 359.81%than that of the control.Gongzhulingmycin could increase the defense enzyme activities of rice seedlings at the four-leaf stage before transplanting.The activities of SOD,POD,PPO and PAL were all increased,especially the SOD and PPO activities increased by 57.18%and 28.53%,respectively.【Conclusion】Proper application of gongzhulingmycin before sowing can decrease the critical temperature of germination,promote the growth of seedlings,significantly improve the seedling quality,increase the expression levels of cold tolerance genes,improve the response speed to low temperature stress and raise defense enzyme activities to stimulate the cold tolerance in rice seedlings.