Presence of a long nuclear-localization signal sequence in homeodomain transcription factor Nkx 1.2

Homeodomains,a 60-amino acid sequence encoded by 180 nucleotides,are highly conserved DNA-binding motifs that are present in a variety of transcription factors in species ranging from yeast to humans.The NKX proteins belong to the homeodomain(HD)-containing transcription factor family.They play vital roles in the regulation of morphogenesis.NKX1-2 is one member of the NKX subfamily.At present,information about its nuclear localization signal(NLS)sequence is limited.We studied the NLS sequence of zebrafish Nkx1.2 by introducing sequence changes such as deletion,mutation,and truncation,and identified an NLS motif(QNRRTKWKKQ)that is localized at the C-terminus of the homeodomain.Moreover,the deletion of two amino acid residues(RR)in this NLS motif prevents Nkx1.2 from entering the nucleus,indicating that the two amino acids are essential for Nkx1.2 nuclear localization.However,the NLS motif alone is unable to target cytoplasmic protein glutathione S-transferase(GST)to the nucleus.An intact homeodomain is necessary for mediating the complete nuclear transport of cytoplasmic protein.Unlike most nuclear import proteins with short NLS sequences,a long NLS is present in zebrafish Nkx1.2.We also demonstrated that the sequences of homeodomain of NKX1.2 are well conserved among different species.This study is informative to verify the function of the NKX1.2 protein.

Immunocytochemical detection of HoxD9 and Pbx1 homeodomain protein expression in Chinese esophageal squamous cell carcinomas

AIM: To evaluate the expression pattern of two novel oncofetal antigens, the HoxD9 and Pbx1 homeoproteins in esophageal squamous cell carcinomas (ESCCs) to determine what role they would play in the carcinogenesis of ESCC.METHODS: We obtained tissue samples of ESCC from 56 patients who underwent esophagectomy but not preoperative chemotherapy or radiotherapy. The diagnosis of ESCC was established and confirmed by staff pathologists. We used a highly sensitive, indirect,immunocytochemical method to detect HoxD9 and PbX1 proteins. We qualitatively and quantitively evaluated cells that exhibited and staining using a light microscope.RESULTS: In all observed carcinoma tissue samples, more than 60% of neoplastic cells stained lightly or strongly for HoxD9, and more than 50% of neoplastic cells stained lightly or strongly for Pbx1.CONCLUSION: Our data suggest that HoxD9 and Pbx1 are inappropriately expressed in most human esophageal squamous cell carcinoma. Understanding the role of Hox genes in esophageal epithelial cell carcinogenesis may not only augment early detection but also offer new avenues for treatment of this disease.

Up-regulation of a homeodomain-leucine zipper gene HD-1 contributes to trichome initiation and development in cotton

Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T_1 locus.The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv.Hai7124 was interrupted by a long terminal repeat(LTR)retrotransposon,while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc.T586.Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family,which mainly regulated epidermis hair development in plants.Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation.Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton,while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development.This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton,but also provides a reference for exploring the different forms of trichome development in plants.

Genome-Wide Identification,Expression Profiling and Protein-Protein Interaction Properties of the BEL-Like Homeodomain Gene Family in Apple

BEL1-like homeodomain(BLH)family proteins are homeodomain transcription factors,which are found ubiquitously in plants and play important roles in regulating meristem and flower development.Although BLH proteins have been reported in some plant species,there is very little information available for plants in the Malus genus(e.g.,apple tree:Malus domestica).In the present study,we identified 19 apple MdBLH genes.Phylogenetic analysis revealed that the MdBLH genes could be divided into five groups.Analysis of gene structure showed that MdBLH gene has four exons,and the third exon was 61 bp in length.Chromosomal location analysis suggested that the MdBLH genes are not distributed uniformly on 12 chromosomes.Eleven MdBLH genes were cloned by RT-PCR,and their expression patterns were also determined.Among them,the expression levels of MdBLH4.1 and MdBLH9.1 could be induced by sodium chloride stress,while the expression levels of MdATH1.1,MdBLH8.1,MdBLH8.3,and MdBLH11.1 were down-regulated by such stress.Transcriptional levels of MdATH1.1 and MdBLH7.2 were down-regulated by mannitol stress.The result of yeast two-hybrid experiment showed that MdBEL1.1 interacted with apple ovate family proteins 6(MdOFP6),and MdBLH3.1 interacted with the MdOFP4,MdOFP6,MdOFP13,and MdOFP16 proteins.Our results provide a strong theoretical basis and a valuable reference for analyzing of the biological functions of MdBLH proteins as transcription factors in apple growth,development,and stress and also for the construction of regulatory networks.

Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA 156 Module by the Homeodomain Proteins PENNYWISE and POUND-FOOLISH in Arabidopsis

The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification. In Arabidopsis, an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTER- BINDING PROTEIN-LIKE （SPL） transcription factors, including SPL3, SPL4, and SPL5, function to specify flowers by activating floral meristem identity genes. During shoot development, SPL3, SPL4, and SPL5 are post-transcriptionally regulated by microRNA156 （miR156）. The photoperiod regulated florigenic signal, FLOWERING LOCUS T （FT）, promotes floral induction, in part by activating SPL3, SPL4, and SPL5. In turn, these SPLs function in parallel with FT to specify flower meristems. Two related BELLl-like homeobox genes PENNYWISE （PNY） and POUND-FOOLISH （PNF） expressed in the shoot apical meristem are absolutely required for the specification of floral meristems. Genetic studies show that the floral specification function of FT depends upon PNYand PNF; however, the interplay between these homeodomain proteins and SPLs is not known. In this manuscript, we show that the photoperiodic floral induction of SPL3, SPL4, and SPL5 is dependent upon PNY and PNE Further, PNY and PNF also control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Lastly, ectopic expres- sion of SPL4 partially rescues the pny pnf non-flower-producing phenotype, while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification. These results suggest that： （1） SPL3, SPL4, and SPL5 function is dependent upon PNY and PNF, or （2） expression of multiple SPLs is required for floral specification in pny pnf plants.

Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment

The Arabidopsis （Arabidopsis thaliana L.） genome encodes for four distinct classes of homeodomain leucinezipper （HD-ZIP） transcription factors （HD-ZIPI to HD-ZIPIV）, which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD- ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZlP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.

Brassica napus L. Homeodomain Leucine-Zipper Gene BnHB6 Responds to Abiotic and Biotic Stresses

A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）. The full-length cDNA of BnHB6 was 1 611 bp and contained a 936-bp open reading frame encoding 311 amino acids. Sequence analysis indicated that BnHB6 belonged to the HD-Zip I subfamily. High-stringency Southern boltting analysis showed that BnHB6 appeared in rape as a single copy but had homologous genes. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analysis revealed that BnHB6 was expressed in several tissues tested under control conditions, but that expression was significantly upregulated in shoots by mannitol, NaCI, cold treatment, anaerobic culture, wounding, H2O2, abscisic acid （ABA）, and salicylic acid （SA） treatments, but not by ultraviolet treatment. Further RT- PCR analysis revealed that BnHB6 was a late-responsive gene, the expression of which was not activated by NaCI, cold treatment, H2O2, ABA, and SA at an early time point （20 min） of treatment in the shoot. However, after a certain period of treatment, the induced expression culminated and then declined until the next peak occurred. Tissue-specific analysis revealed that BnHB6 was expressed at certain levels in the roots, shoots, and flowers, and the roots were found to respond to the osmotic stimuli more rapidly than shoots to increase the expression of BnHB6. The present study implies that BnHB6 plays a positive role as a regulator of biotic and abiotic stresses on growth during seedling establishment.

rhe homeodomain of Eyeless regulates cell ;Irowth and antagonizes the paired domain- Jependent retinal differentiation function

Pax6 and its Drosophila homolog Eyeless （Ey） play essential roles during eye development. Ey/Pax6 con- tains two distinct DNA binding domains, a Paired domain （PD） and a Homeodomain （HD）. While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of differ- ent Ey domains on cell growth and on retinal develop- ment. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.

Out of step： The function of TALE homeodomain transcription factors that regulate shoot meristem maintenance and meristem identity

The indeterminate growth pattern displayed by shoots is mediated by the proper maintenance of the shoot meristem. Meristem maintenance is dependent upon the balance of stem cell perpetuation in the central zone （CZ） and organogenesis in the peripheral zone （PZ）. Although the mechanisms that coordinate CZ and PZ function is not understood, meristem cell fate is likely achieved by the spatial interplay between gene regulatory networks and hormone signaling pathways. During shoot maturation, the identity of the shoot meristem as well as the lateral organs are transformed during the vegetative and reproductive transitions. Studies in model plant systems indicate that three amino acid extension （TALE） homeodomain proteins integrate signaling events that transform the identity of the shoot meristem and establish reproductive patterns of growth. This review will highlight the function of TALE homeodomain transcription factors that regulate shoot meristem cell fate and also function with phase specific regulators to maintain shoot meristem identity.

Upregulated lncRNA PRNT promotes progression and oxaliplatin resistance of colorectal cancer cells by regulating HIPK2 transcription

BACKGROUND Colorectal cancer(CRC)is the third most common cancer and a significant cause of cancer-related mortality globally.Resistance to chemotherapy,especially during CRC treatment,leads to reduced effectiveness of drugs and poor patient outcomes.Long noncoding RNAs(lncRNAs)have been implicated in various pathophysiological processes of tumor cells,including chemotherapy resistance,yet the roles of many lncRNAs in CRC remain unclear.AIM To identify and analyze the lncRNAs involved in oxaliplatin resistance in CRC and to understand the underlying molecular mechanisms influencing this resistance.METHODS Gene Expression Omnibus datasets GSE42387 and GSE30011 were reanalyzed to identify lncRNAs and mRNAs associated with oxaliplatin resistance.Various bioinformatics tools were employed to elucidate molecular mechanisms.The expression levels of lncRNAs and mRNAs were assessed via quantitative reverse transcription-polymerase chain reaction.Functional assays,including MTT,wound healing,and Transwell,were conducted to investigate the functional implications of lncRNA alterations.Interactions between lncRNAs and trans-cription factors were examined using RIP and luciferase reporter assays,while Western blotting was used to confirm downstream pathways.Additionally,a xenograft mouse model was utilized to study the in vivo effects of lncRNAs on chemotherapy resistance.RESULTS LncRNA prion protein testis specific(PRNT)was found to be upregulated in oxaliplatin-resistant CRC cell lines and negatively correlated with homeodomain interacting protein kinase 2(HIPK2)expression.PRNT was demonstrated to sponge transcription factor zinc finger protein 184(ZNF184),which in turn could regulate HIPK2 expression.Altered expression of PRNT influenced CRC cell sensitivity to oxaliplatin,with overexpression leading to decreased sensitivity and decreased expression reducing resistance.Both RIP and luciferase reporter assays indicated that ZNF184 and HIPK2 are targets of PRNT.The PRNT/ZNF184/HIPK2 axis was implicated in promoting CRC progression and oxaliplatin resistance both in vitro and in vivo.CONCLUSION The study concludes that PRNT is upregulated in oxaliplatin-resistant CRC cells and modulates the expression of HIPK2 by sponging ZNF184.This regulatory mechanism enhances CRC progression and resistance to oxaliplatin,positioning PRNT as a promising therapeutic target for CRC patients undergoing oxaliplatin-based chemotherapy.

基于CT图像的活检可帮助预测非小细胞肺癌患者HOPX表达状态和预后的研究

目的本研究旨在阐明具有放射基因组特征的基于CT图像的活检以预测非小细胞肺癌(NSCLC)患者仅同源域蛋白同源盒(homeodomain-only protein homeobox,HOPX)基因表达状态和预后。方法根据HOPX的表达将患者标记为HOPX阴性或阳性,并将其分为训练数据集(n=92)和测试数据集(n=24)。在对116例患者进行基因与图像特征的相关性分析中,从1218个图像特征中选出了8个与HOPX表达相关的显著特征作为放射基因组特征候选,以预测HOPX的表达状态和预后。结果通过叠加集成学习模型建立具有放射基因组特征的影像活检模型,在测试数据集中,模型显示出对HOPX表达的预测能力,ROC曲线下的面积为0.873,Kaplan-Meier曲线的预测能力(P=0.0066)。结论具有放射基因组特征的基于CT图像的活检可以帮助医生预测HOPX在非小细胞肺癌中的表达状况和预后。

3D腹腔镜结肠癌根治手术疗效分析及预后评价

目的探讨3D腹腔镜结肠癌根治手术疗效及安全性,分析Bmi-1和CDX-2表达与结肠癌临床病理特征及预后的关系。方法对比分析腹腔镜手术与开腹手术2组切口总长度、手术时间、术中失血量、清扫淋巴结数目、术后12 h疼痛评分、肠功能恢复时间及术后平均住院日7项指标;采用免疫组化EliVision TM plus两步法检测结肠癌组织Bmi-1和CDX-2的表达,分析其与患者年龄、瘤体大小、分化等级、淋巴结转移及5年内复发转移5项临床病理特征的相关性。结果腹腔镜组与开腹组比较,手术时间、清除淋巴结数目无显著差异,但切口总长度小,手术失血量少,术后12 h疼痛评分低,肠功能恢复快,术后平均住院日短。结肠癌组织Bmi-1和CDX-2阳性率分别为79.1%和64.2%。Bmi-1表达与年龄、瘤体大小、分化等级无关,在有淋巴结转移、5年内复发转移患者中其阳性率显著高于无淋巴结转移、5年内无复发转移者;CDX-2表达与年龄、瘤体大小无关,在低分化等级、有淋巴结转移、5年内复发转移患者中其阳性率显著低于(高+中)分化、无淋巴结转移、5年内无复发转移者。Bmi-1和CDX-2表达呈负相关性。结论3D腹腔镜结肠癌手术具有满意的根治效果和安全性,癌组织Bmi-1高表达、CDX-2低表达预示术后易发生复发转移,患者预后不良。

四维超声联合血清circHIPK3、circ0003416诊断胎儿先天性心脏病价值

目的:探索四维超声联合血清环状RNA同源域相互作用激酶3(circHIPK3)、环状RNA0003416(circ0003416)诊断胎儿先天性心脏病(CHD)价值。方法:收集2021年11月-2023年11月本院接诊的胎儿均疑似CHD孕妇324例临床资料,以产后随访或引产病理解剖结果为金标准,分为CHD组(43例)、非CHD的对照组(281例)。收集两组一般资料,采用实时荧光定量PCR法检测血清circHIPK3、circ0003416水平;记录四维超声检测结果,Kappa行一致性检验;受试者工作特征(ROC)曲线分析四维超声联合血清circHIPK3、circ0003416对CHD的诊断价值。结果:CHD组血清circHIPK3(1.41±0.18)高于对照组(1.19±0.13),血清circ0003416(0.82±0.11)低于对照组(1.03±0.14)(均P<0.05);四维超声诊断CHD与最终确诊结果的一致性较好(Kappa=0.809,P<0.001);四维超声、血清circHIPK3、circ0003416诊断CHD的曲线下面积(AUC)均较高,但联合诊断AUC(0.976)及敏感度(97.7%)最高(P<0.001)。结论:CHD母体血清circHIPK3升高、血清circ0003416水平降低,四维超声联合血清circHIPK3、circ0003416水平对CHD的诊断价值较高,可为CHD临床筛查提供参考。

敲低PHF6对三阴性乳腺癌细胞迁移能力的影响探讨

目的探讨植物同源结构域指蛋白6(PHF6)对三阴性乳腺癌MDA-MB-231细胞迁移能力的影响。方法通过癌症基因组图谱(TCGA)中的数据分析PHF6在各类乳腺癌组织[Luminal型、人表皮生长因子受体-2(HER2)高表达型和三阴性乳腺癌]中的表达情况及其表达对患者生存率的影响。利用siRNA干扰技术在三阴性乳腺癌细胞MDA-MB-231中敲低PHF6,通过划痕实验检测敲低PHF6后细胞迁移能力的变化。采用实时荧光定量逆转录聚合酶链式反应(RT-PCR)实验检测乳腺癌细胞中上皮细胞-间充质转化(EMT)相关基因的表达情况。结果PHF6在各类乳腺癌中(Luminal型、HER2高表达型和三阴性乳腺癌)均出现了明显过表达现象,三阴性乳腺癌中表达最高,且高表达PHF6降低了乳腺癌患者的存活率。用siNC、siPHF6转染MDA-MB-231乳腺癌细胞,24 h后RT-PCR结果显示,与siNC组细胞中PHF6的RNA表达水平(1.033±0.09238)相比,siPHF6组细胞中PHF6的RNA表达水平(0.1054±0.006194)明显下调(P<0.001)。划痕结果显示,与siNC组划痕后6、18、24 h划痕间隙[(433.82±18.28)、(217.87±18.43)、(86.96±39.72)μm]相比,siPHF6组在划痕后6、18、24 h划痕间隙[(639.86±30.63)、(464.25±21.41)、(248.55±20.17)μm]均较大(P<0.001、<0.001、<0.01),愈合较慢。RT-PCR结果显示,与siNC组EMT相关基因CDH2、波形蛋白(VIM)的表达[(1.01±0.056)、(1.01±0.045)]相比,在MDA-MB-231细胞中敲低PHF6,siPHF6组CDH2、VIM表达[(0.25±0.036)、(0.632±0.054)]均降低(P<0.001、<0.001)。结论敲低PHF6能抑制三阴性乳腺癌MDA-MB-231细胞的迁移能力。

HOPX表达与局部晚期乳腺癌新辅助化疗疗效的关系

目的探讨局部晚期乳腺癌组织中同源域蛋白同源盒(HOPX)表达及外周血HOPX甲基化与新辅助化疗疗效的关系。方法回顾性分析2020年1月至2023年10月咸阳市中心医院收治的129例局部晚期乳腺癌患者的资料,年龄(50.69±5.48)岁;中高分化71例、低分化58例;ⅡB期48例、Ⅲ期81例;淋巴结转移87例。根据新辅助化疗疗效分为病理完全缓解(pCR)、非pCR。检测患者新辅助化疗前癌组织、癌旁组织中HOPX表达及外周血HOPX甲基化水平。分析局部晚期乳腺癌患者癌组织中HOPX表达、外周血HOPX甲基化率表达与病理特征关系,影响化疗疗效的因素,癌组织中HOPX mRNA相对表达量、外周血HOPX甲基化率预测局部晚期乳腺癌患者新辅助化疗疗效的价值。采用t检验、χ^(2)检验,影响因素用Cox比例风险模型,受试者操作特征曲线(ROC)评估预测效能。结果癌组织中HOPX mRNA相对表达量高于癌旁组织[(1.97±0.32)比(1.13±0.21),P<0.05]。低分化、Ⅲ期、有淋巴结转移的患者癌组织中HOPX mRNA相对表达量、外周血HOPX甲基化率分别高于中高分化、ⅡB期、无淋巴结转移者[(2.19±0.37)比(1.79±0.32)、(2.23±0.39)比(1.53±0.26)、(2.14±0.35)比(1.62±0.28),81.03%(47/58)比56.34%(40/71)、75.31%(61/81)比54.17%(26/48)、73.56%(64/87)比54.76%(23/42),均P<0.05]。Cox回归分析显示:肿瘤分期(RR=5.557,95%CI:2.286~13.505)、分化程度(RR=5.360,95%CI:2.206~13.027)、HOPX mRNA相对表达量(RR=4.455,95%CI:1.833~10.827)、HOPX甲基化率(RR=4.362,95%CI:1.795~10.602)是影响局部晚期乳腺癌患者新辅助化疗疗效的因素(均P<0.05)。ROC分析结果显示,HOPX mRNA相对表达量联合HOPX甲基化率预测局部晚期乳腺癌患者新辅助化疗疗效的曲线下面积高于单独预测(0.894比0.774比0.768)。结论局部晚期乳腺癌患者癌组织中HOPX mRNA相对表达量、外周血HOPX甲基化率与病理特征、新辅助化疗疗效相关,二者联合预测患者新辅助化疗疗效的效能良好。

大豆GmHAT5的克隆及其转基因百脉根的抗盐分析

【目的】从大豆盐胁迫基因表达谱中筛选并克隆得到同源异型域亮氨酸拉链蛋白(HD-Zip)家族基因GmHAT5,将其转化豆科模式植物百脉根并进一步探究其抗盐调控机制。【方法】使用多种生物信息学软件对GmHAT5的开放读码框(ORF)、编码蛋白的分子量、等电点、序列结构和蛋白定位等进行预测,同时将大豆GmHAT5蛋白与其他10个物种的同源蛋白进行比对分析,并对GmHAT5在大豆不同器官及盐胁迫下的表达特性进行分析。此外,构建GmHAT5的植物超表达载体,通过对发根农杆菌LBA1334的转化,得到"复合体"百脉根植株,并在盐胁迫条件下对其进行抗盐表型分析;通过对根癌农杆菌EHA105的转化,获得百脉根的稳定转化株系,并对其进行抗盐表型分析及相关生理指标检测。【结果】多种生物信息学软件分析表明,该片段包含1个1 038 bp的ORF,编码345个氨基酸。GmHAT5的理论分子量和等电点分别为39.17 k D和4.63。GmHAT5蛋白定位于细胞核,与其他HD-Zip家族蛋白一样,属于典型的核蛋白。序列分析表明,GmHAT5包含一个同源异型结构域和一个亮氨酸拉链结构域,属于第I类同源异型域亮氨酸拉链蛋白。同源蛋白比对表明其与野生大豆GsHAT5同源性最高。基因表达特性分析表明,GmHAT5在大豆植株的各个不同器官中均有表达,是一个受盐诱导上调表达的HD-Zip类转录因子。发状根转化的结果表明,使用200 mmol·L^(-1) NaCl处理植株7 d后,"复合体"植株生长状态良好,对照组植株叶片明显萎蔫、失绿;不同NaCl浓度处理离体转基因发状根14 d后,对照组较试验组发状根明显干枯、生长受到抑制。稳定转化结果显示,不同盐浓度处理14 d后,转GmHAT5百脉根与两组对照植株相比生长状态更好。相关生理指标检测结果显示,与两组对照相比,转基因百脉根植株中丙二醛含量和相对质膜透性明显降低(P<0.05),而叶绿素含量和根系活力则显著升高(P<0.05)。测定不同植株阳离子含量的结果表明,转基因百脉根株系与两组对照相比,Na^+在叶片和根中含量显著下降,K^+和Ca^(2+)在叶片和根中含量显著升高。【结论】从大豆中克隆得到一个编码HD-ZipⅠ类同源异型域亮氨酸拉链蛋白基因GmHAT5,该基因在大豆中受盐胁迫诱导表达量显著升高。超量表达GmHAT5显著增强百脉根的耐盐能力,发状根转化法可以作为一种快速有效筛选抗盐候选基因的手段。推测GmHAT5在大豆盐胁迫应激调控中扮演重要角色。

麦角甾苷调节HIPK2-P53通路对人大肠癌裸鼠移植瘤的治疗作用

目的:研究肉苁蓉提取物麦角甾苷对人大肠癌裸鼠移植瘤模型肿瘤生长的作用,及对人大肠癌细胞HIPK2-P53通路的影响,探讨麦角甾苷抑制人大肠癌的作用机制.方法:通过腋下接种人结肠癌HCT-116细胞,建立人大肠癌裸鼠移植瘤模型,随机分为:模型组(生理盐水)、麦角甾苷低[20mg/(kg·d)]、中[40 mg/(kg·d)]、高[80 mg/(kg·d)]剂量组和5-氟尿嘧啶(5-fluorouracil,5-FU)组[1 mg/(kg·d)],经腹腔分别给药0.2mL,连续给药2 wk.2 wk后处死裸鼠,分别检测裸鼠瘤体体积、瘤质量,采用免疫组织化学法检测瘤组织中凋亡相关蛋白同源结构域相互作用蛋白激酶2(homeodomain-interacting protein kinase 2,HIPK2)、P53、Bax和Bcl-2的表达情况.结果:麦角甾苷低、中、高剂量及5-F U对裸鼠瘤体大小抑制率分别为:48.41%、61.04%、63.75%和75.14%,瘤体质量抑制率分别为:42.79%、53.90%、60.99%、66.19%.免疫组织化学结果显示,裸鼠大肠癌肿瘤组织中麦角甾苷低、中、高剂量及5-FU组HIPK2的表达量分别为:4.83±0.62、8.46±0.99、11.90±1.21、13.50±0.94;P53的表达量分别为:14.59±0.90、17.60±1.40、23.10±2.10、22.44±2.05;B a x表达量分别为14.41±0.38、15.84±0.54、26.28±0.55、26.34±2.33;Bcl-2的表达量分别为14.08±1.04、11.93±0.93、7.48±0.86、5.46±0.67.结论:麦角甾苷能够抑制人大肠癌裸鼠移植瘤生长,上调凋亡相关蛋白HIPK2、P53、Bax的表达,下调Bcl-2表达,其抑制大肠癌的作用与促进肿瘤细胞凋亡密切相关.

大豆再生相关基因GmWUS的克隆及表达分析

WUS基因在植物再生过程中具有重要作用。为初步了解GmWUS的功能,基于已公布的大豆基因组数据对该基因进行了相关生物信息学分析,表明GmWUS和At WUS蛋白在酸性区域存在较大差异,而其他结构域较保守。从大豆茎尖克隆到GmWUSa的全长序列,该基因亚细胞表达定位于细胞核,表明它可能是一种同源异型域转录因子。q PCR检测种子萌发过程中子叶GmWUS的表达变化,发现GmWUSa和GmWUSb表达量均随萌发天数上升,但GmWUSa在后期表达量下降。丛生芽诱导期间,子叶节处GmWUS的表达呈现出一种先增后减的近似周期规律,表明GmWUS可能参与了丛生芽的分化和再生。比较GmWUSa和GmWUSb的表达变化趋势,推测两基因的功能可能在时间上互补。本文的结果有助于研究GmWUS在大豆再生过程的作用,解释丛生芽的形成机制和作为过表达GmWUS基因增强大豆再生能力、提高转化效率的依据。

Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog

Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells.However,the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown.Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family.The rest ofnanog contains no apparent homology to any known proteins characterized so far.It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes.To test this hypothesis,we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs.Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities.Despite the fact that it contains no apparent transactivation motifs,the C-terminal domain is about 7 times as active as the N-terminal one.This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.

神经元素3与成对盒基因4促进胰腺十二指肠同源框蛋白1诱导间充质干细胞向胰腺分泌细胞分化

目的:探索神经元素3(neurogenin 3,NGN3)与成对盒基因4(paired box gene 4,PAX4)对胰腺十二指肠同源框蛋白1(pancreatic and duodenal homeobox factor1,PDX1)驱动间充质干细胞(mesenchymal stem cells,MSCs)向胰腺β细胞分化过程中的作用。方法:构建PDX1基因及NGN3与PAX4双基因表达腺病毒,重组腺病毒Adxsi-CMV-PDX1感染诱导MSCs1周后,再行重组腺病毒Adxsi-CMV-NGN3/CMV-PAX4感染诱导MSCs;分别检测PDX1、PAX4、NGN3、NK转录因子相关的2.2(NK transcription factor related,gene family 2,locus2,NKX2.2)、v-maf muscu-loaponeurotic fibrosarcoma oncogene homolog B(MafB)、胰岛素(Insulin)、胰高血糖素(Glucagon)多种胰腺分泌细胞相关分子表达情况。结果:成功构建腺病毒Adxsi-CMV-PDX1与Adxsi-CMV-NGN3/CMV-PAX4;MSCs经Adxsi-CMV-PDX1与Adxsi-CMV-NGN3/CMV-PAX4分步诱导后,免疫细胞化学与间接荧光检测显示PDX1、NGN3与PAX4因子在细胞核内稳定表达;重组腺病毒稳定转染5 d后,细胞开始变圆并集聚成团,用双硫腙(dithizone,DTZ)染色细胞质呈亮红色;细胞经诱导1周后,用RT-PCR检测到神经源分化因子1(neurogenic differentiation 1,NruroD1)与NKX2.2表达,2周后可检测胰岛素/胰岛素原分子;间接荧光检测显示诱导后的细胞先后开始表达NKX2.2、MafB等转录因子与胰岛素/胰岛素原、胰高血糖等胰岛分泌细胞相关分子,但未能检测到v-maf musculoaponeurotic fibro-sarcoma oncogene homolog A(MafA)与C肽分子表达。结论:NGN3与PAX4对PDX1诱导间充质干细胞向胰腺分泌细胞分化具有促进作用。