围脂滴蛋白3(Perilipin 3,PLIN3)是围脂滴蛋白家族成员之一,已被证实与脂滴初生相关,主要负责甘油三酯(Triglyceride,TAG)向脂滴的掺入.奶牛乳腺是乳脂合成旺盛器官,PLIN3在泌乳乳腺中表达,但其是否参与乳腺中乳脂合成尚不明确.采用基因过表达及Western blot等方法检测PLIN3对奶牛乳腺上皮细胞中TAG含量的影响,通过添加乳脂合成前体物以及抑制剂方法研究mTOR信号通路对PLIN3表达的调节.结果表明,PLIN3过表达极显著提高奶牛乳腺上皮细胞中TAG含量(P<0.01).短链脂肪酸乙酸和β-羟基丁酸激活mTOR信号通路,提高细胞中PLIN3表达;采用雷帕霉素抑制mTOR信号通路激活则显著抑制短链脂肪酸对PLIN3表达的诱导作用(P<0.05).该结果提示PLIN3表达与奶牛乳腺上皮细胞中乳脂合成相关,乙酸和β-羟基丁酸通过激活mTOR信号通路调节PLIN3表达,促进乳脂合成.

溶血磷脂酸酰基转移酶(Sn-1-acylglycerol-3-phosphate O-acyltransferase 6,AGPAT6)是脂类物质合成代谢相关酶,但其在奶牛乳脂合成中调节作用尚不明确.研究采用荧光定量PCR、Western blot等方法检测奶牛泌乳循环过程中乳腺组织中AGPAT6表达变化、奶牛乳腺上皮细胞中AGPAT6对乳脂合成影响以及相关信号通路分子表达变化.结果表明,AGPAT6 mRNA和蛋白质表达在泌乳期奶牛乳腺组织中极显著高于青春期和干奶期(P<0.01).在具有乳脂合成能力的奶牛乳腺上皮细胞中,AGPAT6基因过表达极显著提高细胞中TAG含量,AGPAT6基因干扰获得相反结果.AGPAT6过表达可激活PI3K-AKT-mTOR信号通路,添加PI3K抑制剂会抑制AGPAT6诱导PI3K-AKT-mTOR信号通路激活及细胞中TAG合成(P<0.01).以上结果显示AGPAT6表达与泌乳奶牛乳脂合成呈正相关,AGPAT6通过激活PI3K-AKT-mTOR信号通路促进奶牛乳腺上皮细胞中乳脂合成.

为了研究4F2hc在奶牛乳腺中的表达模式及调控方式,进一步明确氨基酸在奶牛乳腺上皮细胞中的跨膜转运过程,本研究采用Western blotting和实时荧光定量PCR技术检测了4F2hc在泌乳期和干奶期奶牛乳腺组织中的表达变化;在体外培养的泌乳期奶牛乳腺上皮细胞中添加亮氨酸,采用Western blotting和实时荧光定量PCR技术检测其对奶牛乳腺上皮细胞中4F2hc表达的影响;采用雷帕霉素抑制剂抑制mTOR信号通路,使用Western blotting方法检测mTOR信号抑制后奶牛乳腺上皮细胞中4F2hc表达以及乳蛋白合成的变化.结果 显示,在泌乳期的奶牛乳腺组织中4F2hc的mRNA和蛋白表达水平均显著或极显著高于干奶期(P＜0.05,P＜0.01);在体外培养的奶牛乳腺上皮细胞中添加亮氨酸可以极显著提高乳腺上皮细胞中4F2hc的mRNA和蛋白质表达水平(P＜0.01);亮氨酸刺激可以激活细胞内的mTOR信号通路(P＜0.05),而雷帕霉素处理则可以显著抑制mTOR信号分子的磷酸化并极显著抑制亮氨酸诱导的4F2hc的表达(P＜0.05,P＜0.01),进而极显著抑制β-Casein的合成(P＜0.01).以上研究结果表明,4F2hc基因的表达与奶牛乳腺的泌乳活性之间呈正相关,亮氨酸可以通过激活mTOR信号通路来调节4F2hc基因的表达,进而影响乳蛋白的合成.

The investigation was performed in Stalin Park of Harbin city.Greenbelt and tourist in the park were investigated by first-hand investigation and random interview.The urban waterfront attributes such as publicity and miscibility,and present conditions of landscape elements of Stalin Park such as landform,vegetation and architecture were summarized.And then evaluated the expressive way and problems of these attributes and gave out suggestions such as the naturalization of river revetment,strengthening service management and valuing detailed design.

The chlorophyll fluorescence parameters of cold-resistant cultivar,Dongnongdongmai 1,and CK,Jimai 22,were measured during cold acclimation and frozen periods.The results showed that maximum fluorescence yield(Fm),maximum quantum yield of PS II(Fv/Fm),actual quantum yield(yield),photochemical quenching(qP)in new leaf of CK Jimai 22 was significantly higher than that of Dongnongdongmai 1 in the cold acclimation periods.After the low temperature acclimation,these parameters of Jimai 22 declined rapidly,and were significantly lower than that of Dongnongdongmai 1.The changing trend of Fo during cold acclimation and frozen periods was as Fo in the new leaf of Dongnongdongmai 1 was stable and its reduction in Dongnongdongmai 1 and Jimai 22 were 26.2% and 52.8%,respectively.The fluorescence parameters tendency in old leaf of Dongnongdongmai 1 was consistent with that of Jimai 22,these fluorescence parameters in old leaf of Dongnongdongmai 1 were significantly higher than that of Jimai 22 after cold acclimation period.Chlorophyll fluorescence parameters can be used to identify the cold resistance of wheat cultivars.The identification stage should be at the end of cold acclimation and the beginning of frozen period.The test position of wheat seedling should be the new leaf of cultivar.

To analyze the distinctions in adaption during cold acclimation period among the cultivars which were different in the cold resistance, the physiological traits in the leaves and tiller nods of two kinds of winter wheat cultivars during the cold acclimation period were observed and measured. The results showed that the electrical conductivity of the cold resistant cultivar Dongnong Dongmai1 was lower than that of Jimai22; the content of proline and the soluble protein of both cultivars increased during the cold acclimation period, and the content of proline and the soluble protein in leaves and tiller nods of the Dongnong Dongmai1 was higher than that of Jimai22.

The changes of the fresh weight,dry weight,relative water content,electrical conductivity and the content of proline between the two cultivars Dongnong Dongmai1 and Jimai22 during the cold acclimation period were compared to analyze the physiological basis in winter hardiness of winter wheat.The results showed that the dry weight of Dongnong Dongmai1 was higher than that of Jimai22 during the cold acclimation period,when the temperature was lower than 0 ℃,the dry weight of Dongnong Dongmai1 still increased,but Jimai22 decreased.The relative water content of Jimai22 higher than that of Dongnong Dongmai1,the relative water content of Dongnong Dongmai1 decreased rapidly at October 29,and the relative water content of Jimai22 decreased rapidly at November 2.The content of proline in tiller nods of Dongnong Dongmai1 was higher than that of Jimai22.The relative electrical conductivity of Jimai22 was higher than that of Dongnong Dongmai1 during cold acclimation period.

The seedlings with 6 leaves of winter wheat cultivar "Dongnongdongmai 1" were treated in 5 combinations of temperature/ days based on temperature data for low temperature acclimation and over-winter period of winter wheat in 3 years,to reveal the change of proteins expressed in winter wheat cultivar "Dongnongdongmai 1" under different low temperature treatments.The changes of proteins in the seedling stems under soil surface were observed in different temperature treatments,and the results showed that there were significant changes in 17 protein spots by 4 low temperature treatments and 8 reappearing spots of those proteins were picked up for spectrum analysis.The 8 proteins were attributed to the processes of 26S protein dehydration,respiration,tricarboxylic acid cycle,chlorophyll synthesis,glyoxylate cycle,respectively,and of which one spot was cold resistant protein.Some speculations on the frozen resistance of the cultivar was proposed preliminarily by the amount changes and functions of the 8 proteins under different low temperature degrees.

The paper discussed the breeding strategy for yield breakthrough of spring wheat in Heilongjiang Province from the following 5 aspects. The aim was to solve the problem of the slow increasing of yield in spring wheat cultivars, the lower interest of planting wheat comparing with other crops, and the decline of spring wheat planting area in Heilongjiang Province, which arise the irrational structure of crop cultivation. From the yield components, genetic improvement of increasing kernel number in spring wheat should be prominently enhanced on breeding, which based on high harvest amount of spikes; the anti-lodging ability of wheat cultivars must be strengthened to avoid the risk of lodging as the weight of spikes increase; the yield breakthrough of spring wheat rested with enhancing the dry matter accumulation amount; coordinating the conflict on high and stable yield with yield and quality in the genetic improvement of wheat yield.

Protein extracting method,protein dissolution,protein loading amount and gel strip transfer were tested for the protein extracted from seedling rhizome of winter wheat cultivar Dongnong Winter Wheat No.1.The results showed that the loss of low abundance protein in TCA/acetone protein extracting process(T process) was less than that in urea/thiourea protein extracting process(N process) and the spots in protein map in T process were more than that in N process.The purer protein dissolved two times in the hydrated liquid could maintain higher voltage of 8000 volt by Isoelectric Focusing and Electrophoresis.The protein loading of 10mg by two times dissolution in hydrated liquid could obtain clear map with more protein spots and better separation effect.After adding 400μl 0.3% common agarose solution to glue surface and washing support film of gel strip with 200μl electric buffer,the gel strips could be transferred easily to second dimension glue surface and no air bubbles appeared between gel strips and glue surface.