【作者】 袁陵；

【导师】 李正国；

【作者基本信息】 重庆大学 ， 生物学， 2010， 硕士

【摘要】 奉节脐橙(Citrus sinensis Osbeck)是一个优良的甜橙鲜食品种,在国内外市场享有较高声誉,是重庆市柑橘出口中唯一具有较强竞争优势的品种。但奉节脐橙果皮褐变发生较严重,主要症状为油胞破裂下陷、形成褐斑,极大地影响了果实的外观质量和商品价值。目前对脐橙果皮褐变的发生机制尚未进行深入研究,缺乏相应的控制措施,这已成为目前脐橙商品生产发展的关键制约因素。因此迫切需要开展对奉节脐橙果皮褐变的生理及分子机制研究。本研究以‘奉园72-1’脐橙为实验材料,果实采后分别采用清水、50 mg.kg-1 2,4-D+250 mg.L-1施保功、1%氯化钙+50 mg.kg-1 2,4-D+250 mg.L-1施保功、2%氯化钙+50 mg.kg-1 2,4-D+250 mg.L-1施保功、1%硝酸钙+50 mg.kg-1 2,4-D+250 mg.L-1施保功、2%硝酸钙+50 mg.kg-1 2,4-D+250 mg.L-1施保功等对果实进行10 min浸钙处理,晾干后用PE薄膜袋单果包装,置于20℃常温下贮藏,定期取样观察果实的褐变情况并测定生理指标,包括褐变指数、相对电导率、丙二醛含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、多酚氧化酶(PPO)活性。并对钙处理后对果实品质的影响进行了分析,定期测量了失重率、可溶性固形物、可滴定酸、维生素C含量等。利用荧光定量PCR方法,检测了对照组和钙处理组CsCAB基因和它的两个靶蛋白基因CsGAD、CsATPase在果皮褐变进程中的表达水平的变化。获得的主要研究结果如下:①钙处理可有效降低果皮褐变指数。1% Ca(NO3)2处理组贮藏末期褐变指数比对照低20%,2% Ca(NO3)2处理组贮藏末期褐变指数比对照低15%。CaCl2处理效果优于Ca(NO3)2处理,1% CaCl2处理效果最好,贮藏110 d后褐变指数为25%,比对照降低了27.5%,降低果皮褐变指数能力为1% CaCl2>2% CaCl2>1% Ca(NO3)2>2% Ca(NO3)2。②钙处理显著降低了脐橙果实的相对电导率。随着贮藏时间的延长,对照组果实的相对电导率上升速率明显高于浸钙果实。结果显示,1% CaCl2处理效果最好,贮藏110 d后相对电导率比同期对照低13%,能显著抑制果皮相对电导率的升高。③与对照相比,各处理都能不同程度地减少丙二醛的积累。其中1% CaCl2、2% CaCl2处理效果最佳:在贮藏末期,1% CaCl2处理的果实丙二醛含量为9.35 nmol.g-1,比对照低6.32 nmol.g-1,2% CaCl2处理的果实丙二醛含量为10.16 nmol.g-1,比对照低5.51 nmol.g-1。1% Ca(NO3)2、2% Ca(NO3)2处理的样品中丙二醛含量分别比同期对照低23.4%和11.3%。④钙处理可以明显提高奉节脐橙果实贮藏过程中SOD的活性,其中以1% CaCl2处理的果实中SOD活性最高,110 d时比同期对照高120 U.g-1。贮藏期间,对照处理POD活性都低于钙处理,其中1% CaCl2处理前40 d活性最高,40 d以后处于缓慢下降,但还是显著高于对照组的POD活性。Sporgon+ 2,4-D、2% CaCl2、1% Ca(NO3)2和2% Ca(NO3)2处理在贮藏50 d后POD活性都趋于平缓,但都显著高于对照组活性。在贮藏40 d时,1% Ca(NO3)2、1% CaCl2、2% CaCl2、和Sporgon+ 2,4-D处理,都导致PPO活性下降,与对照组差异极显著,其中1% CaCl2处理的PPO活性值最低,比对照组中PPO活性低了77%,且与1% Ca(NO3)2、2% CaCl2和Sporgon+ 2,4-D处理相比,差异显著。贮藏的不同时期,1% CaCl2处理PPO活性都低于其他处理,也低于对照组。⑤钙处理果实贮藏期间,对品质无不良影响。贮藏20 d后,1% CaCl2处理组其失重率均显著小于对照组;1% CaCl2处理组的果实的可溶性固形物含量在第40 d达到高峰,其含量在20~110 d均显著高于对照;1% CaCl2处理组在30 d和50 d时,其可滴定酸的含量显著高于对照,到第110 d时,各处理与对照组基本一致;2% CaCl2处理后的果实的维生素C含量在20 d时达到高峰,50 d后,维生素C含量高于同期对照组,但不显著,110 d时,维生素C含量显著高于对照。另外在110 d时,各处理组含量均显著高于对照。⑥利用荧光定量PCR技术分析钙处理后褐变进程中的钙调蛋白基因CsCAB及其靶蛋白基因CsGAD和CsATPase表达情况,结果显示,1% CaCl2处理组中的CsCAB基因表达量显著高于对照组果实中的表达。贮藏末期100 d时,1% CaCl2处理组CsCAB基因的表达水平是对照组的3倍。CsGAD基因在1% CaCl2处理组表达水平有所波动,但都高于对照组,贮藏50 d时表达水平最高。随着贮藏时间的延长,对照组和钙处理组的CsATPase基因都出现下调表达。贮藏100 d后,1% CaCl2处理组中的CsATPase基因表达量最低。更多还原

【Abstract】 ‘Fengyuan 72-1’navel orange (Citrus sinensis Osbeck) is welcomed by consumers for its good tasty and beautiful color. But it’s prone to develop peel pitting, which has been described as a severe disorder with characteristics of the extensive collapsed areas of the flavedo (outer colored part of the peel) and part of the albedo (inner part of the peel) that becomes brown with time. The external quality and consequently the market value of the fruit are decreased by peel pitting.The navel orange fruits were obtained from local orchards, and treated with different concentrations of CaC12 and Ca(NO3)2 in this study.Six sets of fruits were dipped in water (the control), 50 mg.kg-1 2,4-D+ 250 mg.L-1 Sporgon,1% CaCl2+ 50 mg.kg-1 2,4-D+ 250 mg.L-1 Sporgon, 2% CaCl2+ 50 mg.kg-1 2,4-D+ 250 mg.L-1 Sporgon, 1% Ca(NO3)2+ 50 mg.kg-1 2,4-D+ 250 mg.L-1 Sporgon, 2% Ca(NO3)2+ 50 mg.kg-1 2,4-D+ 250 mg.L-1 Sporgon for 10 min respectively. After drying in air, the fruits were bagged and stored at room temperature. Physiological and biochemical indicator correlated incidence of peel pitting including pitting index , relative electric conductivity,malondialdehyde (MDA) content,activities of Polyphenol Oxidase (POD), Polyphenol Oxidase (PPO), Superoxide Dismutase (SOD) were measured every ten days. The fruit quality such as weight loss, soluble solid content , titrtable acidity, Vc content,were also determined. Finally, this study used fluorescence quantitative PCR to measure the changing expression of CsCAB and its two target protein genes CsGAD, CsATPase during the period of peel pitting in control and calcium treated groups respectively.The main results of the research were as follows:①Calcium treatments could effectively reduce the peel pitting. The peel pitting index of 1% Ca(NO3)2 at the end of storage was 20% lower than the control while 2% Ca(NO3)2 was 15% lower than the control. CaCl2 treatments were more effective than Ca(NO3)2 treatments, and the peel pitting index of 1% CaCl2 treatment after 110 days of the storage was 25%, which was 27.5% lower than the control. The capacity of reducing peel pitting index was 1% CaCl2> 2% CaCl2> 1% Ca(NO3)2> 2% Ca(NO3)2.②Calcium treatments significantly reduced relative conductivity of navel orange fruits. With the proloning of the storage time, the increasing rate of relative conductivity of the control fruit was obviously higher than that of calcium treatments. The results showed that, 1% CaCl2 treatment was the most efficient, the relative conductivity of which was 13% lower than control, and could significantly inhibit the increase of relative conductivity.③Different calcium treatments could minimize the accumulation of MDA in varying degree compared with the control. Among these treatments, the effects of 1% CaCl2, 2% CaCl2 treatments were the best: at the end of storage, MDA content of 1% CaCl2 treatment fruits was 9.35 nmol.g-1, which was 6.32 nmol.g-1 lower than control, while that of 2% CaCl2 treatment was 10.16 nmol.g-1, which was 5.51 nmol.g-1 lower than control. MDA content of 1% Ca(NO3)2 and 2% Ca(NO3)2 treated samples were respectively 23.4% and 11.3% lower than control.④Calcium treatments significantly increased the SOD activity of‘Fengjie’navel orange fruits during storage, of which 1% CaCl2 treatment was the highest, 120 U.g-1 higher than the control after 110 days of the storage. During storage, POD activity of the control was lower than calcium treatments, of which 1% CaCl2 treatment was the highest before 40 days , later in a slight decline, but still significantly higher than that of control. POD activities of Sporgon+2,4-D treatment, 2% CaCl2 treatment, 1% Ca(NO3)2 treatment, 2% Ca(NO3)2 treatment after 50 days of storage became leveling off, which were significantly higher than the control.After 40 days of storage, 1% Ca(NO3)2, 1% CaCl2, 2% CaCl2, and Sporgon+2,4-D treatment, had led to PPO decreasing significantly compared with the control group, of which PPO activity in 1% CaCl2 group was the lowest, 77% lower than the control, and with significant difference compared with 1% Ca(NO3)2, 2% CaCl2, and Sporgon+2,4-D groups. In different periods of storage, PPO activities of 1% CaCl2 treatment were always lower than any other treatments, including the control group.⑤The fruit quality of navel oranges treated with calcium did not decline during the whole storage period. After 20 days of storage, the weight loss of 1% CaCl2 treatment group was significantly lower than control group; the soluble solid content of 1% CaCl2 treatment group reached a peak at the 40th day, and its content during the 20 ~ 110 days of storage were significantly higher than control; titratable acid content of 1% CaCl2 treatment group was significantly higher than the control at the 30th day and 50th day. At the 110th day, each treatment is almost consistent with the control group; Vc content of 2% CaCl2 treatment reached a peak at the 20th day while 50 days later , higher than the control group, not significantly, but at the110th day, much higher than the control. Also at the 110th day, each treatment group was significantly higher than the control.⑥We used fluorescence quantitative PCR technique to study CsCAB, the calmodulin in citrus, and its target protein gene CsGAD and CsATPase gene expression during the period of browning in samples with calcium treatments and control. The results showed that, CsCAB gene expression in 1% CaCl2 treatment was significantly higher than the expression in the control. At the 100th day of storage , CsCAB gene expression of 1% CaCl2 treatment group was 3 times more than the control group. Although CsGAD gene expression levels appeared to fluctuate, but were always richer than the expression of the control group, with the highest expression level at the 50th day of storage. CsATPase gene expression reflected a downward trend both in the calcium treatments and the control. After 100 days of storage, CsATPase gene expression of 1% CaCl2 treatment group was the lowest.更多还原