【作者】 许耀照；

【导师】 孙万仓；

【作者基本信息】 甘肃农业大学 ， 作物学（作物遗传育种）， 2020， 博士

【摘要】 低温是限制农作物地理分布和生长发育的环境因子之一,研究作物抗寒机理,对培育抗寒作物新品种,促进农业生产有重要的价值。白菜型冬油菜（Brassica rapa L.,AA）为西北地区唯一的冬季油料作物,适应寒旱环境,含有丰富的抗寒基因。本研究以白菜型冬油菜强抗寒品种陇油7号和弱抗寒品种天油4号为研究材料,低温胁迫下,通过叶片组织结构观察、生理生化指标分析、蛋白组学和microRNA测序分析以及PsbR基因克隆,从生理、蛋白和RNA水平研究白菜型冬油菜抗寒生理基础及分子机理,主要研究结果如下:1.白菜型冬油菜对低温胁迫的生理生化响应叶片气孔和组织结构观察发现,低温条件下冬油菜叶片气孔密度、气孔面积、气孔周长、栅栏组织和海绵组织厚度均变小,叶片变薄,细胞间隙变大,而强抗寒品种陇油7号的气孔和组织结构变化较弱抗寒品种天油4号更明显;光合生理分析发现,低温条件下,陇油7号叶片P_n、F_v/F_m、F_v/F_o、PI、叶绿素荧光诱导动力学曲线O-J-I-P的F_o（O相）、F_k（K相）、F_j（J相）、F_m（P相）以及荧光参数F_o-k、F_k-j、F_i-P和F_j-i均较天油4号明显降低;进一步生理生化研究发现,低温胁迫后,陇油7号可溶性蛋白含量和CAT酶活性均低于天油4号,而其MDA含量高于天油4号;结合光合指标的变化趋势,说明陇油7号叶片细胞膜系统和PSII受损以及光抑制程度均较天油4号严重,其地上部表现较强的低温敏感性。根部生理生化指标分析发现,低温胁迫后,陇油7号根中EL明显小于天油4号,而其根中可溶性蛋白和可溶性糖含量以及SOD酶活性均明显高于天油4号,说明根中可溶性蛋白和可溶糖的积累以及较强SOD酶活性增强白菜型冬油菜低温耐受性。低温条件下,陇油7号叶中IAA、ZR、ABA含量、（IAA+ZR+GA₃）/ABA值和总多酚含量均较天油4号低,而根中ABA和总多酚含量均较天油4号高。相关性分析表明越冬率与冬油菜内源激素IAA、ZR和GA₃含量呈负相关,而与根中ABA和总多酚含量呈正相关,说明ABA和总多酚含量对冬油菜的低温耐受力非常重要。2.白菜型冬油菜低温诱导的蛋白组学分析通过iTRAQ测序分析,从-4℃低温胁迫的陇油7号和天油4号叶中鉴定出142个差异富集蛋白（DAPs）,其中29个DAPs丰度上调,113个DAPs丰度下调,这些DAPs参与了10个代谢通路;从根中鉴定出70个DAPs丰度上调和104个DAPs丰度下调,这些DAPs参与了8个代谢通路。功能分析得出,白菜型冬油菜叶片通过减弱TCA循环和PPP途径,降低乙醛酸盐和二羧酸盐的能量代谢,削弱光合作用,平衡叶中糖类物质的含量来适应低温逆境;而冬油菜根通过蛋白质合成、根细胞壁变厚、根细胞木质化程度增加、降低根中能量代谢（磷酸戊糖途径和氧化磷酸化）、抑制糖消耗、削弱碳水化合物代谢、使可溶性糖积累来响应低温逆境。3.白菜型冬油菜低温胁迫响应的microRNAs分析采用高通量测序技术从低温胁迫的陇油7号和天油4号叶中鉴定出2个保守的差异表达miRNAs（miR166e-3p和miR166h-3p）,根中鉴定出4个保守的差异表达miRNAs（miR166e-3p,miR319e-1,miR319a,miR319-2）,这些保守的miRNAs均属于miR166和miR319家族;另外,从根中鉴定出4个新的miRNAs（Bra-Novel-m3153-5p,Bra-Novel-m0894-3p,Bra-Novel-m3936-5p,Bra-Novel-m0040-3p）,推测miR166、miR319和新的miRNAs在白菜型冬油菜响应低温胁迫中发挥重要作用。miRNA靶基因预测和功能分析发现,miR319的靶基因TCP4和MYB104参与次生细胞壁合成,推测miR319及其靶基因可能调控冬油菜根部次生细胞壁合成而响应低温胁迫。4.白菜型冬油菜PsbR基因的克隆及低温胁迫下表达分析采用DDRT-PCR和5′RACE技术从陇油7号叶中克隆到PsbR（Photosystem II subunit R）基因,该基因全长为570 bp,编码包含417 bp的一个完整开放阅读框ORF（Open reading frame）,其编码138个氨基酸,该基因编码的蛋白属于PsbR超家族,与甘蓝、山嵛菜和芥菜型油菜的PsbR蛋白具有较近的亲缘关系。实时荧光定量PCR分析发现,低温胁迫后,PsbR相对表达量下降,说明低温抑制PsbR基因的表达。结合低温胁迫后陇油7号P_n和F_v/F_m降低的趋势,推测陇油7号通过下调PsbR在低温条件下的表达来调控光合系统,使其P_n和F_v/F_m下降,增强光抑制程度来响应低温胁迫。更多还原

【Abstract】 Low temperature is one of the environmental factors that limited the distribution,growth and development of crops.Therefore,the study on cold-tolerant mechanism of crop has extensively applicated value for breeding of cold-tolerant variety.Winter turnip rape（Brassica rapa L.）is a only winter oil crop in northwestern China for its adaption to cold and dry environment and is rich in many effective cold tolerance genes.In this study,we used the cold tolerant winter turnip rape variety Longyou 7 and cold sensitive variety Tianyou 4 as experiment material.Leaf anatomic structure,physiological-biochemical index the photosynthetic responses,proteomics and microRNA sequencing and cloning of PsbR gene were determined under the condition of low temperature,which revealed the response mechanism of low temperature from the level of psychology,protein and RNA.The results were summarized as follows:1.Physiological and biochemical responses to low temperature stress in B.rapa.Stomatal traits and leaf anatomic structure were observed and showed that stomatal density,stomatal area and total stomatal circumference per unit area in two cultivars decreased.Moreover,leaf thickness,palisade tissue and sponge tissue thickness became thinner and cellular space grew bigger under low temperature.Compared with Tianyou 4,Longyou 7 had smaller stomatal density,stomatal area,total stomatal circumference per unit area and leaf thickness.The parameters of photosynthesis and fluorescence were analyzed and showed that photosynthetic rate（P_n）,maximum photochemical efficiency（F_v/F_m）,the potential activity of PSⅡ（F_v/F_o）,photosynthetic performance index（PI）,F_o（O phase）,F_k（K phase）,F_j（J phase）and F_m（P phase）of chlorophyll fluorescence induced kinetics curves of O-J-I-P as well as oxygen-evolving complex ativity state(F_o-k),Q_A reduction ability(F_k-j),Q_B slow reduction PQ activity(F_i-P)and Q_B（including fast reduction PQ）reduction ability(F_j-i)of leaves in Longyou 7 decreased significantly more than those in Tianyou 4.Further studies on the physiological and biochemical showed thatthe soluble protein content and CAT activity of Tianyou 4 were higher than those of Longyou 7 and the MDA content of the former was lower than that of the latter under cold stress.Combined with the variation tendency of photosynthetic parameters,damage to cell membranes and photosystemII as well as photosynthetic inhibition of Longyou 7 were more serious than thoese of Tianyou 4.These results indicated that leaves of‘Longyou 7’have higher sensitivity to cold stress than do those of Tianyou 4.The physiological and biochemical indexes in roots were studied and showed that EL level of Longyou 7 was significantly lower that of Tianyou 4,but other physiological indexes（soluble protein,soluble sugar and superoxide）of‘Longyou 7’were higher than those of Tianyou 4.These results indicated the roots of Longyou 7 with higher tolerance to freezing stress than those of Tianyou 4.The contents of endogenous hormones（IAA、ZR、GA₃ and ABA）and total polyphenols were sampled to analyze.The results showed that compared with Longyou 7,the contents of IAA、ZR、ABA and total polyphenols as well as ratio of（IAA+ZR+GA₃）/ABA in Tianyou 4 leaves were higher,but contents of ABA and total polyphenols in its roots were lower under low temperature.The correlation analysis showed that overwint-ering rate of winter turnip rape had negative correlation with contents of IAA、ZR and GA₃ in leaves and roots under lower temperature,but had positive correlation with contents of ABA and total polyphenols of roots.These results indicated that contents of ABA and total polyphenols are very important for cold tolerance of winter turnip rape.2.Proteomics profiling induced by low temperature in B.rapaUsing iTRAQ technology,142 differently accumulated proteins（DAPs）were identified between cold-stressed leaves of Longyou 7 and Tianyou 4,and these DAPs participated in10 pathways.174 DAPs were identified between roots of two varieties,and these DAPs participated in 8 pathways.Based on the functional analysis,we concluded that decreased energy metabolism via the TCA cycle,PPP,glyoxylate and dicarboxylate,together with decreased photosynthesis,enabled leaves of winter turnip rape to balance synthesis and consumption of sugar.This adaptive balance permitted the survival of winter turnip rape under low temperature stress.We also concluded that increased ribosome and biosynthesis of secondary metabolites,decreased energy and carbohydrate metabolism via PPP,OXPHOS,fructose and mannose metabolism could improve protein synthesis,cell wall thickness of root and accumulation of sugar,which are conducive to survival of winter turnip rape under freezing stress.3.Analysis of low temperature stress responsive microRNAs in B.rapaTwo conserved miRNAs（miR166e-3p and miR166h-3p）and four conserved miRNAs（miR166e-3p,miR319e-1,miR319a,miR319-2）were identified from the leaves and root of two low-temperature-stressed winter turnip rape varieties.These conserved miRNAs belonged to members of miR166 and miR319 families.Morever,four novel miRNAs（Bra-Novel-m3153-5p,Bra-Novel-m0894-3p,Bra-Novel-m3936-5p,Bra-Novel-m0040-3p）were identified from the root of two low-temperature-stressed winter turnip rape varieties.It was speculated that miR166,miR319 and these novel miRNAs play an important role in winter turnip rape response to cold stress.Prediction of miRNA and functional analysis were found that TCP4 and MYB104 are target genes of miR319,which involved in secondary cell wall synthesis,indicating miR319 and the target genes might respond to cold stress by regulating secondary cell wall synthesis.4.Cloning and expression analysis of PsbR gene in B.rapa under Low temperature stressThe PsbR was separated and cloned from the leaf by differential display reverse transcription PCR（DDRT-PCR）and rapid-amplification of cDNA ends（RACE）.The full length cDNA of PsbR is 570 bp and the open reading frame（ORF）encoding a protein of138 amino acids,was 417 bp.PsbR belongs to the PsbR superfamily,which presented high similarity with PsbR proteins from B.oleracea var.Oleracea,E.salsugineum and B.juncea.The relative expression of PsbR gene under low temperature stress went down.Combined with decreased net photosynthetic rate（P_n）and efficiency of primary conversion of light energy of photosystemⅡ（F_v/F_m）,we speculated the expression of the PsbR gene may be inhibited under low temperature stress,and then decreased net photosynthetic rate and photochemical efficiency of PSⅡ.更多还原