协同抑制番茄ACO1对果实成熟及病程相关蛋白基因表达的影响

 【目的】探讨协同抑制番茄ACO1基因对果实成熟和病程相关蛋白基因表达、内源乙烯生物合成及果实耐贮性的影响。【方法】采用PCR或RT-PCR方法克隆了番茄ACC氧化酶1、ACC氧化酶3、EBF1、PR1、PR5以及NP24基因片段，并以此制备探针，以协同抑制ACO1的转基因番茄和野生型番茄为研究对象，进行Northern杂交，同时测定了伤害叶片和果实的乙烯释放量，并进行了果实贮藏试验等。【结果】Northern杂交结果表明，番茄ACO1基因表达被抑制后，与果实成熟相关基因LeACO3和LeEBF1，以及病程相关蛋白基因LePR1、LePR5 和LeNP24的表达量急剧降低。乙烯释放量测定试验和果实贮藏试验结果表明，协同抑制LeACO1番茄完整和受伤叶片以及完整果实内源乙烯释放量相对于野生型番茄大大减少，成熟果实贮藏时间延长。【结论】协同抑制番茄ACO1基因表达的同时，与果实成熟相关基因和病程相关蛋白基因的表达也不同程度地受到抑制，而且其内源乙烯生物合成减少，果实耐贮性增强。     

反义ACS转基因乙烯缺陷型番茄的生理特性

反义 ACC合成酶 (ACS)基因番茄的采后生理性状与普通番茄不同 ,该番茄的果实和叶片乙烯以及果实呼吸强度受到抑制 ,果实乙烯释放量为 0 ,没有出现呼吸高峰 ,呼吸强度在 10～ 14 mg.kg-1.h-1之间波动 ,极显著低于对照 (高峰时为 2 4 .55mg.kg-1.h-1)。反义番茄的叶绿素降解和番茄红素的合成亦受阻 ,果实在采后和贮藏期间逐渐变为黄色或桔黄色而番茄红素的合成很少 ,到采后 30 d仅有 0 .0 4 A.g-1(fw)。乙烯催熟采后 30 d的反义番茄 ,番茄红素增加 6 0倍。反义番茄 PG酶活性在采后缓慢上升 ,到采收后的第 4 5天达到最高为 4 5.16μmol.g-1.h-1,但其活性也仅为普通番茄的一半。乙烯利催熟前后 ,反义番茄的 PG酶活性没有显著性变化     

反义LeEIL2基因表达载体的构建及在番茄中的转化

为了研究LeEIL2基因在番茄果实成熟过程中的作用 ,将全长的番茄LeEIL2基因以反义方向构建到植物表达载体 pBI1 2 1上 ,经过酶切鉴定正确后 ,将该载体转入农杆菌EHA1 0 5中 ,通过农杆菌介导法转化番茄 ,经组织培养和抗性筛选获得再生植株 ,PCR检测再生植株 ,初步鉴定为阳性植株 ,为获得转反义LeEIL2番茄果实打下基础。     

耐贮藏转基因番茄F2代植株乙烯代谢与细胞膜透性的研究

研究乙烯对番茄野生型幼苗及转反义NR、F1'、F2、F3乙烯基因型番茄突变体叶片电解质外渗率以及对番茄生理代谢的影响.结果表明:随着乙烯浓度的增加,野生型番茄丽春、中疏4号、中疏6号幼苗叶片黄化、衰老和脱落,甚至死亡,细胞膜透性也增大,乙烯对中疏4号番茄的膜透性影响最大,丽春次之,对中疏6号影响最小.转反义F1'、F2、F3乙烯基因型番茄突变体叶片的膜透性极显著地小于野生型,而转反义NR突变体番茄叶片的膜透性与野生型差异不明显.说明转反义F1'、F2、F3基因在叶片上的表达被抑制后,膜代谢发生了明显的改变,膜透性降低,植物对乙烯的反应降低,从而延迟了果实的成熟,植株衰老减缓.     

转反义ACC合成酶基因番茄与普通番茄果实植物内源激素含量的变化

 转反义 ACS( ACC合成酶基因 )番茄与普通番茄果实激素平衡方式不同。普通丽春番茄果实采前 IAA、Z+ ZR(玉米素 +玉米素核苷 )逐渐下降 ,GA在花后 40 d出现高峰 ,之后逐渐下降 ;ABA呈递增趋势 ;IAA/ ABA花后 2 0 d最高 ,花后 40 d至果实变色期变化不显著 ,但果实粉红期至红色期 IAA/ ABA显著下降。转反义 ACS番茄表现了与丽春番茄不同的变化趋势 ,其IAA/ ABA从花后 2 0 d至绿熟期呈上升趋势 ,而绿熟期至腐败期缓慢下降。转反义 ACS番茄生长类激素的含量在果实生长发育时期 (绿熟期之前 )与普通番茄没有显著差异 ,但在成熟衰老时期显著地高于普通番茄。     

转ACS番茄采后脂质过氧化相关酶研究初报

乙烯是控制成熟衰老的重要激素。研究表明,在乙烯的生物合成途径Met、SAM、ACC、Ethylene中,催化SAM～ACC的ACC合成酶（ACS）是乙烯生物合成的限速酶。我们利用反义基因技术将ACC合成酶基因反向插入番茄,得到了乙烯合成缺陷型的转基因番茄。这种番茄果实的成熟衰老可以大大延迟,在室温下放置3个月不变红,而催熟后表现出与一般番茄相同的成熟性状。本试验以转ACS基因番茄果实作为试验材料,研究了在乙烯合成受阻的情况下膜质过氧化产物MDA和相关酶的变化。研究表明：SOD（超氧化物歧化酶）、CAT（过氧化氨酶）、POD（过…     

番茄采后成熟过程种子和果皮中脱落酸与乙烯代谢的关系

以番茄中杂101为材料，研究采后果实成熟过程种子和果皮中脱落酸（ABA）含量与乙烯生物合成的关系，以及外源ABA及其生物合成抑制剂（fluridone）处理对果实ABA含量和乙烯释放量的影响。结果表明：采后番茄果实成熟过程中．种子的乙烯释放量、1-氨基环丙烷-1-羧酸（ACC）含量和ABA含量均高于同时期果皮；种子和果皮中ABA和ACC含量的峰值都出现在乙烯跃变之前；ACC氧化酶（ACO）活性变化趋势与ABA含量及乙烯释放量的变化趋势相一致；外源ABA处理使果皮和种子中ABA含量显著增加，促进了果实乙烯的生成；fluridone处理则相反。以上证据表明，番茄果实中的ABA通过刺激乙烯的生成促进果实成熟，种子可能通过调控果实内ABA含量和乙烯释放量而影响果实的成熟。     

利用反义技术和RNA干扰技术改良番茄品质特性的研究

综述了利用反义技术和RNA干扰技术,通过抑制番茄果实发育过程中许多重要基因(如PG基因、ACC合成酶基因、ACC氧化酶基因、LeCOP1LIKE、TBG6等)的表达,使番茄的耐贮性、番茄红素含量和裂果率等品质特性发生显著变化的研究进程,试验证明,反义技术和RNA干扰技术是抑制靶基因表达的有效手段。     

转FRO2基因番茄突变体的抗缺铁黄化特性

为了增加植物的抗缺铁黄化能力,研究了转FRO2基因番茄突变体抗黄化特性.结果显示FRO2基因转化番茄后,使生长在缺铁胁迫下的植株根系铁还原酶活性大大增强(转FRO2基因番茄植株根系酶活性是对照2倍),番茄铁吸收能力和有效铁还原能力大大加强,植株中有效铁含量增加(转FRO2基因番茄植株叶片有效铁含量是对照的1.5倍),转FRO2基因番茄的植株叶片颜色显著比对照绿,黄化程度减轻,黄化指数降低(对照的黄化指数是转FRO2基因番茄的1.3倍),植株生长量显著高于对照(转基因植株株高高于对照50%).在正常条件下,转FRO2基因番茄根系的FRO2基因表达量增加,铁还原酶活性增加了1.75倍,转FRO2基因番茄植株叶片有效铁含量是对照植株的2倍,是其他耐贮藏转基因番茄的(转反义NR、ACC、CTR1基因)2～3.5倍以上.     

不同转基因番茄GalUR的表达与Vc含量

为了得到高维生素C(Vc)含量新品种, 克隆和构建了GalUR半乳糖醛酸还原酶基因, 并转入了番茄. 通过不同转基因突变体GalUR基因表达和Vc含量分析发现: GalUR 基因在转GalUR 基因番茄中得到了过量表达; 在转FRO2-铁还原酶基因的番茄突变体中表达量增加了60%; 在耐贮藏转基因番茄F3、 F2、 F1中也均高于对照, 其中F3的表达量最高, 是对照的2～3倍; 在非转基因中蔬4号和6号的表达量高于丽春番茄1倍; 所有供试GalUR 基因表达量均为红果最多、粉红果次之, 青果最少. 转基因番茄果实Vc含量变化与上述GalUR基因的表达有相同规律. 转GalUR3基因番茄果实和叶片Vc含量高于对照2倍以上. GalUR基因的表达还与乙烯合成酶基因ACS、 CTR1和铁还原酶FRO2基因的表达有关.     

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Ⅰ on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase I on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato (Lycopersicon esculentum) were cloned, such as the 1-aminocyclopropane-1-carboxylic acid oxidase 1 gene (LeACO1), 1-aminocyclopropane-1-carboxylic acid oxidase 3 gene (LeAC03), EIN3-binding F-box 1 gene (LeEBF1), pathogenesisrelated protein 1 gene (LePR1), pathogenesis-related protein 5 gene (LePR5), and pathogenesis-related protein osmotin precursor gene (LeNP24) by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig (AC++) and the LeACO1 co-suppression tomatoes (V1187 and T4B), respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carried out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.     

Effects of Ca2+/CaM Messenger System Inhibitors on Ethylene-Induced Increase in Lycopene Content

The changes of lycopene content during ripening and senescence of tomato fruit and the relationship between ethylene glycol-bis (EGTA, Ca2+ chelator), verapamil (Vp, Ca2+ channel blockers), trifluoperazine (TFP), chloropromaize (CPZ) (CaM antagonism) and ethylene-induced increase in lycopene content in tomato fruit were investigated. Lycopene content accumulated obviously during ripening and senescence of tomato fruit after harvest at pink stage. Low temperature inhibited but ethylene enhanced the lycopene content.Meanwhile, ethylene also promoted calmodulin (CaM) content in tomato fruit, which was related to the concentration of ethylene. When EGTA, Vp, TFP and CPZ with ethylene were used to treat tomato fruit, ethylene-induced increase in lycopene content could be reversed, indicating that blocking Ca2+ channel in plasma membrane or chelating extracellular Ca2+ or inhibiting the activity of CaM could decrease the action of ethylene, and suggesting that Ca2+-CaM messenger system may be involved in lycopene increase inducedby ethylene.     

Changes of Activities in NAD Kinase and NADP Phosphatase During Ripening and Senescence of Tomato and Strawberry Fruits

Activities of NAD kinase(NADK)and NADP phosphatase and relationship between the two enzymes and temperature, respiration, ethylene production and trifluoperazine(TFP) were studied during ripening and senescence of strawberry and tomato frnits after harvest at 4℃and 20℃. The activity of NAD kinase in strawberry decreased slowly during first four days, then increased gradually. The NADP phosphatase activity increased at the second day, decreased the next day,then increased again. In tomato fruit, the activities of NAD kinase and NADP phosphatase increased at the second day, decreased with the ripening and senescence of the fruit. The change trend of NAD kinase and respiration in the two fruits were similar, the same were NADP phosphatase and ethylene production. TFP enhanced the activity of NAD kinase and had little effect on NADP phosphatase. Low temperature(4℃ ) activated the NAD kinase and reduced the activity of NADP phosphatase. These results indicated that the NAD kinase and NADP phosphatase were related to the ripening and senescence of strawberry and tomato fruits. The activation of NAD kinase probably postponed the ripening and senescence of the fruits.     

ABA对番木瓜成熟软化的影响及其与乙烯的关系

研究ABA对番木瓜果实成熟软化的影响及其与乙烯的关系,结果表明：番木瓜果实采后乙烯产率逐渐上升,在第8天达到高峰,硬度迅速下降。外源ABA和乙烯利处理促进了果实乙烯的释放以及果实硬度的下降,可溶性固形物含量的上升和果面颜色变化,加速了果实成熟软化进程。ABA生物合成抑制剂NDGA处理则抑制了乙烯的产量,果面颜色变化和硬度的下降及可溶性固形物含量的上升。此外,外加Co2＋和1-MCP处理可抑制ABA对乙烯产率、果实硬度下降,可溶性固形物上升和果面颜色变化的促进作用。ABA对番木瓜果实成熟的影响可能主要是通过刺激乙烯的产生进行的。     

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Ⅰ on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase Ⅰ on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l-aminocyclopropane-1-carboxylic acid oxidase 1 gene （LeAC01）, 1- aminocyclopropane-l-carboxylic acid oxidase 3 gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5）, and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig （AC＋＋） and the LeAC01 co-suppression tomatoes （V1187 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carded out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.     

不同剂量短波紫外线照射对采后番茄后熟和发病的影响

给采后绿熟番茄照射不同剂量短波紫外线并人工接种交链孢霉，测定其呼吸和乙烯产生速率，特色及发病指标，进行了贮藏保鲜效果分析，结果表明，2．4－3．6kJ.m^-2照射剂量能推迟呼吸及乙烯释放高峰1－3d，延缓番茄的成熟和转色，控制贮藏病害发生。