Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Construction and Partial Biological Characteristics Analysis of Listeriolysin S llsB Gene Deletion Strain

In order to analyze the effect of listeriolysin S (LLS) llsB gene deletion on the biological characteristics of Listeria monocytogenes (LM),this study used homologous recombination to construct the llsB gene deletion strain LM90-ΔllsB,and the biological characteristics of growth characteristics,median lethal dose (LD₅₀) and organ-borne bacteria were studied in healthy Kunming male mice at 8 weeks old and weighing 40 g±5 g.The llsB gene deletion strain was successfully constructed,and the deletion strain had good genetic stability through continuous passage to 20 generations in vitro.Based on its growth curve examination,we found that the growth rate of the mutant strain was slightly higher than that of the parent strain.The results of mice infection test showed that the LD₅₀ of the parent strain and the deletion strain were 10^6.17 and 10^6.50 CFU,respectively.Compared with the parent strain,the amount of bacteria load of the deletion strain in the liver and spleen of the mice was extremely significantly decreased (P<0.01),the results showed that the infection ability of the mutant strain on mice was obviously weakened.No Listeria monocytogenes was detected in the brain.The results suggested that llsB gene might have direct or indirect regulatory effect on some biological characteristics of LM90,and it would provide a theoretical basis for further understanding of the pathogenic mechanism of LLS,prevention and control of listeriosis.     

不同类型水稻品种产量和氮素吸收利用对大气CO2浓度升高响应的差异

目的 探明不同类型水稻品种产量和氮素吸收利用对FACE(大气CO₂浓度增高)响应的差异。方法 以常规粳稻、杂交籼稻、常规籼稻共6个品种为供试材料,研究FACE对不同类型水稻产量、氮素吸收利用的影响。结果 1）FACE处理极显著提高了水稻产量,平均增加24.17%, 常规籼稻增幅最大,FACE和对照均以杂交籼稻最高;2）FACE处理显著增加了单位面积穗数,常规粳稻增幅最大,并显著增加了杂交籼稻和常规籼稻每穗粒数;3）FACE处理显著提高了成熟期吸氮量和氮素籽粒生产效率,成熟期吸氮量平均增加21.23%,杂交籼稻增幅最大, FACE和对照均以常规籼稻最高;氮素籽粒生产效率平均增加7.33%,杂交籼稻增幅最大,FACE和对照均以杂交籼稻最高。成熟期吸氮量对产量促进作用略大于成熟期氮素籽粒生产效率;4）FACE处理降低了植株含氮率,成熟期平均下降0.105个百分点,常规粳稻降幅最大。FACE处理极显著提高植株干物质量,成熟期平均增加23.95%,常规籼稻增幅最大;FACE处理显著提高常规籼稻和杂交籼稻成熟期单穗吸氮量,分别增加10.79%、13.93%,但常规粳稻下降了9.60%;FACE处理显著提高了成熟期群体吸氮强度,平均增加22.29%,杂交籼稻增幅最大。FACE处理对水稻全生育期天数无显著影响;FACE处理显著提高茎鞘、叶片、穗各器官吸氮量,叶片增幅最大,平均增加51.86%,杂交籼稻增幅最大;FACE处理显著提高了不同生育阶段吸氮量,抽穗-成熟阶段增幅最大,平均增加108.90%,杂交籼稻增幅最大;5）植株干物质量、单穗吸氮量、吸氮强度、穗吸氮量、抽穗-成熟阶段吸氮量对成熟期总吸氮量的促进作用分别大于植株含氮率、单位面积穗数、生育天数、茎鞘叶吸氮量、移栽-分蘖和分蘖-抽穗阶段吸氮量;6）FACE处理显著提高了氮肥偏生产力,降低了每百千克籽粒需氮量,前者平均增加24.16%,常规籼稻增加最多;后者平均降低4.7%,常规籼稻降幅最大。结论 FACE处理可显著提高水稻产量和氮素吸收利用效率,但品种间差异较大。     

Wx^mp基因背景下可溶性淀粉合成酶基因SSⅡa和去分支酶基因PUL对水稻蒸煮食味品质的影响

【目的】分析在同一主效基因(Wx^mp)背景下可溶性淀粉合成酶基因SSⅡa和去分支酶基因PUL对稻米蒸煮食味品质的影响,以期为水稻品质遗传改良提供依据。【方法】选择在SSⅡa和PUL存在多态性而其他淀粉合成酶相关基因没有多态性的半糯品系宁0145和粳稻品种武运粳21进行杂交,获得F2群体与F3株系。利用分子标记,选择含有Wx^mp基因的F2单株与F3株系,将这些F2单株与F3株系分成SSⅡa^nPUL^n、SSⅡa^nPUL^w、SSⅡa^wPUL^n和SSⅡa^wPUL^w4种基因型(n和w分别表示该基因来源于宁0145和武运粳21),分析不同基因型蒸煮食味品质性状的差异,探讨同一Wxmp基因背景下不同SSⅡa和PUL等位基因对蒸煮食味品质性状的影响。【结果】不同基因型间蒸煮食味品质性状均存在显著差异,来源于武运粳21的SSⅡa^w基因和PUL^w基因分别使直链淀粉含量增加0.29%~1.00%和0.62%~1.18%,且PUL的效应大于SSⅡa,两者间存在互作效应。SSⅡa^w基因和PUL^w基因降低胶稠度和崩解值,提高了热浆黏度、冷胶黏度、消减值和回复值,对糊化温度、峰值黏度和峰值时间的作用较小。【结论】明确了Wx^mp背景下SSⅡa和PUL基因对稻米蒸煮食味品质的遗传效应,该研究结果为SSⅡa和PUL基因的分子标记辅助选择改良稻米品质提供了理论依据。     

江苏近年育成粳稻新品种/系的稻瘟病抗性基因及穗颈瘟抗性分析

【目的】明确水稻品种携带的抗稻瘟病基育种应用价值,是利用抗病基因培育抗病品种控制病害流行的重要前期工作。【方法】利用功能标记分析了14个抗稻瘟病基因在江苏近年育成的195个粳稻新品种/系中的分布情况,并对其中158个品种和17份携带抗稻瘟病基因Pigm的高世代回交株系进行穗颈瘟接种鉴定。【结果】大多数品种携带2~5个抗病基因,但所有品种均不含有Pigm基因;Pib、Pita和Pikh基因在供试品种中的分布频率较高,均在45%以上,其余基因均在30%以内;Pid3、Pid2、Pia、Pb1在新育成品种中的出现频率明显高于审定品种。测试品种对穗颈瘟的抗性主要与3个基因显著相关,贡献率由高至低依次为Pia、Pi3/5/i和Pita;其中,Pia或Pi3/5/i与Pita间的聚合效应均显著高于各基因单独存在时的抗病效应,且以Pia和Pita间的聚合效应最强,携带该基因组合的所有品种对穗颈瘟均表现抗至高抗水平抗性。回交导入Pigm基因的所有17份株系对穗颈瘟的抗性均显著高于各自轮回亲本,且均达到了抗病以上水平。【结论】抗病基因Pigm及基因组合“Pia+Pita”在江苏粳稻抗穗颈瘟育种中具有重要的育种应用价值。     

水稻蜡质稀少突变体wax1的鉴定及基因定位

【目的】 蜡质是植物表面的一种重要保护物质,筛选和鉴定水稻蜡质相关突变体有助于解析水稻蜡质形成的遗传机制。【方法】利用EMS诱变籼稻品种湘早籼6号,从突变体库中筛选出一个蜡质稀少的突变体wax1,考查突变体的形态特征和农艺性状,利用突变体与02428杂交的F₂群体定位目标基因,并通过荧光定量PCR分析相关基因的表达情况。【结果】与野生型相比,wax1突变体具有叶片短小皱褶、叶表面蜡质晶体减少、穗长变短等形态特征;在农艺性状方面,突变体的株高、每穗总粒数和千粒重显著降低,但有效穗数显著高于野生型;遗传分析表明,wax1的突变表型受一对隐性核基因控制,将WAX1基因定位在第10染色体上SSR标记RM5806与InDel标记P1之间,物理距离约为49.8 kb;定位区间内的测序结果表明,突变体中β-酮脂酰-CoA合酶的编码基因发生单碱基突变,导致催化活性中心的一个氨基酸发生改变。WAX1基因的突变显著提高了同源异型盒基因OSH6的表达,同时引起部分IAA基因的差异表达。【结论】WAX1基因突变引起叶表面蜡质晶体的减少,同时通过影响茎尖分生组织和生长素的信号转导引起植株生长发育等多方面的异常。     

金刚砂辅助农杆菌进行玉米基因转化研究

对超声波处理方式进行改进,在超声波处理时,加入金刚砂以辅助EPSPS基因转导到玉米幼胚中。结果表明,800粒处理种子播种出苗93株,玉米苗经大田glyphosate筛选有27株具有抗性,进行PCR电泳检测,显示23株阳性,初步证明EPSPS基因已经转化到玉米植株中,转化率为29%。金刚砂辅助处理玉米基因转化具有可行性,利于生产实际操作。     

豆科全基因组DGAT 基因家族的鉴定与进化分析

本文运用生物信息学方法在12个豆科植物基因组中对DGAT（甘油二酯酰基转移酶）基因进行了全基因组鉴定，发现在四倍体花生和大豆基因组中含有的基因拷贝数相对较多，分别为17、10，这可能是导致其油脂合成能力高于其他作物的重要因素。通过对家族基因进行系统发育分析，发现DGAT 基因在真双子叶植物分化之前已存在，在豆科植物中经历反复的全基因组加倍，使得家族得到扩张；并且串联重复对于家族扩增起到了不可忽视的作用，如葡萄中的两个旁系同源基因，由于串联重复使得基因拷贝数量提高了150%。大豆中DGAT 基因在不同的组织中表达模式与其系统发育关系也表现出了关联性。该基因家族相对保守，与豆科基因组进化基本保持了一致的进化速率。本研究对于认识豆科植物DGAT 基因进化过程具有重要的理论意义，同时在基因组学水平为大豆、花生等油脂合成品质改良提供了理论支撑。     

Dynamics of nitrogen translocation from mature leaves to new shoots and related gene expression during spring shoots development in tea plants (Camellia sinensis L.)

The contribution of N remobilization is crucial for new shoots growth and quality formation during spring tea shoots development. However, the translocation mechanism of N from source leaves to sink young shoots is not well understood. In the present study, ¹⁵N urea was applied to mature tea leaves one week before bud break to track N remobilization in a field experiment. The dynamic changes in plant ¹⁵N abundance, contents of amino acids, and the expression levels of genes related to N metabolism and translocation were followed during the 18‐d development of new spring shoots until three expanding young leaves. The results showed that during the growth of new shoots the amount of ¹⁵N in the shoots increased, whereas the N_dff (N derived from ¹⁵N‐urea) in mature leaves decreased, showing that the foliar‐applied N in mature leaves was readily exported to new shoots. This process was found to be accompanied by decline of chlorophylls. In the mature leaves, expression CsATG18a and CsSAG12 involved in autophagy was dramatically induced (> 4‐fold) at approximately nine days after the bud breaking. The genes involved in the transformation of amino acids, including primarily CsGDH2, CsGDH4, CsGLT3, CsGS1;3, and CsASN2 were upregulated by > 3‐fold after bud breaking. The expression levels of CsATG8A, CsATG9, CsSAG12, CsGS1;1, CsGDH1, and CsAAP6 correlated negatively with the N_dff in mature leaves, but positively with ¹⁵N amount and total N amount in new shoots, suggesting these genes played important roles in N export from mature leaves. In the new shoots, the expression of most genes showed two defined peaks, one on six days and one on 12 days after bud breaking. The expression of CsGS2, CsASN3, CsGLT1, and CsAAP4 positively correlated with the ¹⁵N amount and total N amount in new shoots. These genes might be involved in the transport and re‐assimilation of N from mature leaves. The overall results demonstrated that the translocation of ¹⁵N from mature leaves to new spring shoots was regulated by the genes involved in autophagy, protein degradation, amino acid transformation and transport.     

不同温度下TMV侵染枯斑三生烟的LncRNA差异表达

【目的】 筛选不同温度下烟草花叶病毒（Tobacco mosaic virus,TMV）侵染后枯斑三生烟（Nicotiana tabacum var. Samsun NN）差异表达的长链非编码RNA（long non-coding RNA,lncRNA）,研究lncRNA在枯斑三生烟抗性反应中的作用。【方法】 N基因的温度敏感性使枯斑三生烟在25℃时具备对TMV的抗性、在31℃抗性丧失,在这两个温度条件下对枯斑三生烟接种TMV和磷酸盐缓冲盐水（phosphate buffered saline,PBS）,48 h后提取系统叶总RNA,构建链特异性文库后进行深度测序。对测序结果进行过滤后利用HTSeq将有效数据与近缘品种TN90（N. tabacum var. TN90）基因组比对,筛选得到lncRNA后利用FPKM法估计lncRNA的表达水平。通过edgeR筛选差异表达lncRNA（differentially expressed lncRNA,DElncRNA）,并利用qRT-PCR技术对这一结果进行验证。通过共定位及共表达分析预测DElncRNA的靶基因,通过参考基因组注释、GO和KEGG富集分析研究靶基因的功能。【结果】 4个处理共12个样本经lncRNA-seq各测得约8 000万条clean reads,共获得4 737条已知lncRNA、40 169条新lncRNA。其中64个lncRNA在不同温度条件下TMV侵染后存在差异表达,qRT-PCR测定结果显示这些lncRNA的测序正确率在80%左右,表明本研究所得测序数据具备较高的可信度。对DElncRNA进行靶基因预测,发现一些基因同时被25℃下调和31℃上调的DElncRNA靶向。靶基因注释功能丰富,主要参与植物抗病、激素和代谢等生理过程。部分可能与激素通路相关的lncRNA,在25℃下TMV侵染时呈现下调趋势,而在31℃下TMV侵染则呈现上调趋势。GO富集分析显示靶基因主要参与构成膜、囊泡等组分,具备钙、钾离子通道抑制剂活性等分子功能,使相应离子得以转运引发随后的反应,同时也参与发病、抗原加工和呈现、细胞分裂素代谢等生理过程。KEGG分析发现靶基因显著富集在植物激素信号转导通路,25℃下调和31℃上调的DElncRNA靶基因同时富集在激素信号传导、ABC运输蛋白、苯丙烷类生物合成等通路。【结论】 不同温度（25℃和31℃）条件下TMV侵染枯斑三生烟后,长链非编码RNA差异表达,DElncRNA通过作用于激素信号传导、物质转运等过程参与寄主系统获得性抗性反应。研究结果可为揭示植物系统获得性抗性中lncRNA的调控功能以及新型抗病毒技术开发提供依据。