Maize (Zea mays) is an important food crop, feed source and industrial raw material. With the development of bio-technology, transgenic and other new technologies are widely used in maize breeding. Cauliflower mosaic virus 35S (CaMV 35S) promoter and its enhancer are widely used in transgenic operation of maize and other crops. Therefore, studying the mechanism of CaMV 35S enhancer has important theoretical value for future application. Two trait mutants were screened from a maize activation-tagging mutant library previously constructed by the research group on genetic improvement of maize. Based on the identification of artificial transposon insertion sites in the chromosomes, the spatio-temporal variation of gene RNA expression in the range of about 100 kb upstream and downstream of the transposon insertion sites was analyzed using qPCR. The results showed that the enhancer of CaMV 35S significantly up-regulated the gene expression of adjacent genes (P<0.05), and the transcriptionally increased genes were distributed upstream and downstream of the transposon insertion site, with activation distance of up to 63.7 kb. Of the 4 genes up-regulated expression, the enhancement was dose-dependent, and the expression of the homozygous mutant was significantly higher than that of the heterozygous mutant (P<0.05). Although the CaMV 35S enhancer increased the transcription level of genes in the maize mutants, it did not significantly change the expression pattern of genes in different tissues. The scope, dosage effect, and tissue specificity of CaMV 35S enhancer in maize provide an experimental basis for subsequent use in maize molecular biology, and genetic engineering, and can also provide reference for the application of the promoter in other plants.

在丘陵、山区等有坡度地势栽植苗木时,为了能够保证树苗竖直生长,基于曲柄滑块机构设计了一种可根据地势坡度自动调整挖坑角度的翻转装置.为使自动翻转装置具备较好的工作性能,计算推导了翻转机构中连杆翻转角度与滑块位移的关系式,并采用机械系统动力学自动分析(Automatic Dynamic Analysis of Mechanical Systems,ADAMS)软件对曲柄滑块机构进行运动仿真.该结构提高了苗木种植机的工作性能和智能化水平,同时为种植机的结构设计提供了理论指导.

通过添加Zr元素的方式提高车身用烧结Al-5Si铝合金的综合性能,采用微观组织测试和慢应变速率拉伸(SSRT)试验等方式对其进行组织和力学性能分析.结果表明:Zr可以抑制再结晶过程,使再结晶晶粒尺寸减小.在Al-5Si-Zr板材内形成了更多的黑色第二相颗粒,再结晶晶粒数量也明显增加.板材内都存在弥散分布第二相组织,具有明显的针形结构,添加Zr之后其数量减少.Zr可以对Al-5Si-Zr板材的淬火空位产生钉扎作用,形成更少的可动空位,使晶界相难以发生形核.板材在NaCl溶液内中相对于空气环境达到了更低拉伸强度与伸长率.添加Zr后,空气中板材拉伸强度从346 MPa减小至321.5 MPa,同时伸长率从原先的12.2％减小至11.41％.Al-5Si-Zr板材断口部位形成了密集分布的韧窝,发生了穿晶韧断的结果,断裂尺寸与深度都发生了降低.

为了弥补液压机中传动电驱液压泵引起的节流与溢流损失,提出了一种变转速混合动力液压系统控制方式.系统配备蓄能器后可实现电液电机的能量回收,达到系统泄漏的补偿作用,以及对液压执行器发挥预压紧的功能,可以精确调控运行速率与位置.在Simulation X软件中构建了仿真模型,并从动态特性和能效特性两个方面展开分析.研究结果表明:2.5 s后两电机均发生正向旋转,从而得到总压力;2.7 s时系统压力达到9.1 MPa;介于2.7～6.0 s之间时,压力稳定变化;6.6 s时,总压力信号减小至0.该液压系统表现出了优异的运行特性,形成了平稳的速度曲线.电动机驱动过程中形成了稳定的功率变化,以液压-电气方式进行能量回收时,系统达到了最高的能量回收率,表现出最优的节能效果.开展实验测试可知,该系统可以精确定位,速度曲线运行平稳,基本无冲击,整个系统运行特性良好.

构建植物过表达载体pHZM1 N-P ZmMRP-1::ZmMRP-1,利用农杆菌介导的玉米愈伤组织转化法将其导入玉米自交系A188中,获得12个独立的PCR阳性转化株系.对转基因株系进行Southern blot检测、qRT-PCR分析,发现OE-8、OE-19株系以单拷贝形式插入玉米基因组中并过量表达.对获得的转ZmMRP-1基因T2代材料进行玉米产量相关性状考察,相比较野生型,转基因株系在粒长、粒宽、百粒质量等方面均显著提高,可有效改良玉米产量相关性状,为高产转基因玉米新品种培育提供基础材料支持.

针对工件经磨料流加工后表面的粗糙度数值改变问题,在总结磨料去毛刺机理的基础上,建立粗糙度数学模型,理论分析临界粗糙度的存在条件.通过加工圆管和叶片泵转子槽底孔去毛刺试验分析可知,当上道工序的加工表面的粗糙度值大于该临界粗糙度时,磨料流加工后粗糙度将进一步减小;当上道工序的加工表面的粗糙度值小于该临界粗糙度时,磨料流加工后粗糙度反而增大;最终的表面粗糙度值接近临界表面粗糙度.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

The morphology, whole molecular size distribution and chain-length distribution of maize leaf starch have been characterized and compared to its endosperm starch, to better understand differences between leaf and endosperm starch structure, and the relationship with the functions of starch in these organs. Leaf starch is found to have amylopectin with much shorter chains (virtually none with a degree of polymerization, DP, above 70) than the endosperm amylopectin, which has significant numbers of chains with DP up to ∼120, and has much smaller molecular size (and is present at a much lower amount) than endosperm starch. It is postulated that these pronounced differences arise from the distinct starch synthesis pathways in these organs, and are consistent with the starches' distinct botanical functions: short-term storage requiring relatively rapid degradation for leaf starch, and high crystallinity and high energy density requiring slow degradation for endosperm starch.

The lepidopteran Ostrinia furnacalis is one of the most serious pests of maize production. The Cry1C proteins are group of Bacillus thuringiensis (Bt) proteins that are toxic to the intestine of insects. Overexpression of Cry1C protein has led to increased resistance to lepidopteran pests in several crops. In the present study, the synthetic cry1C* gene that was previously tested in rice was introduced into maize Hi-II genotypes via biolistic gun-mediated transformation. A total of nine independent putative callus were obtained and 87 transgenic plants were positive with cry1C* according to polymerase chain reaction (PCR) analysis. Three highly insect-resistant transgenic plants, ZmKc-2-3, ZmKc-3-2, and ZmKc-3-5, were further confirmed by PCR analysis, field assessment, and genomic southern blotting in the T3 generations. Insect bioassays were conducted in both the field and the laboratory, and showed that progeny of the three transgenic lines were significantly resistant to lepidopteran maize pests during the whole development and growth period. The stable integration and expression of the cry1C* in the three transgenic plants’ progeny were confirmed by reverse transcription-PCR (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA) methods. Hybrids were produced by crossing transgenic line ZmKc-2-3 with the elite inbred line Zheng58. There was small variation among the hybrids and backcross offspring, indicating that these cry1C* transgenic lines can be used to produce insect-resistant hybrids and served as insect-resistant sources for the development of Bt maize.

Phosphomannose isomerase(PMI) gene from Saccharomyces cerevisiae was cloned by PCR,and transformed into Arabidopsis via floral dip method.The seeds were collected and germinated on selecting MS medium which mannose concentration was 1 g/L.Four positive plants were obtained.PCR for genome DNA and RT-PCR for RNA proved the PMI gene had integrated into the genome of Arabidopsis and was transcribed.The intron of yeast pmi was removed successfully in Arabidopsis.GUS activity also was detected in the transgenic plants.All the results means PMI gene from yeast can be used as a selectable marker in Arabidopsis transformation.

以玉米自交系CML295、CML304和18-599R的成熟胚为外植体,结合幼胚离体培养方法,探讨并优化了成熟胚来源的胚性愈伤组织诱导及继代培养方法.对其愈伤组织的形态和组织切片的研究结果显示,继代过程可产生良好的Ⅱ型胚性愈伤组织.在分化培养中分别获得68.6%、75.4%和84.8%的高频再生率,每愈伤组织块成苗数分别为2.45、2.43和2.75.利用基因枪法转化pCAMBIA1301质粒后的GUS瞬时表达效率分别为57.9%、62.5%和73.1%,转化pCAMBIA1303质粒后检测GFP的瞬时表达效率分别为23.3%、40%和45.5%.以上3种基因型成熟胚来源的愈伤组织转化率与其对应的幼胚来源的胚性愈伤组织转化率相似.这一技术体系为玉米的遗传改良和功能基因组研究提供了重要的技术平台.

Using normal maize and o2 maize as materials,the four colorimetric methods of ninhydrin,acid orange-12 dye binding lysine(DBL),2-Chloro-3,5-dinitropyridine(CDNP) and 2,4,6-Trinitrobenzene sulfonic acid(TNBS) were compared and discussed in the regression equation of calibration curve,the lysine content determined by means of these methods,the assay principles and the laboratorial work.The results showed that the colorimetry of 2-Chloro-3,5-dinitropyridine is relatively precise,easy operating,middle costing and less time consuming,especially applicable to the assay of lysine content of families in segregating population from a combination of normal type line and high-lysine line(particularly one with modifier genes).

DEAD-box RNA helicases are involved in various aspects of RNA metabolism, including RNA transcription, premRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation and RNA decay. According to maize S-Mo17 Rf3Rf3 cDNA microarray data, an RNA helicases was found to be upregulated in the later stage of pollen development. Full-length cDNA of this RNA helicases, named ZmRH2 was cloned (GenBank accession number: DQ327709) in maize S-Mo17 Rf3Rf3 pollen using RACE. cDNA sequencing indicated: the full length of ZmRH2 cDNA is 1 652 bp, which contained an open reading frame encoding 407 amino acids from 163th to 1 386 th positions. The encoding amino acid sequence of ZmRH2 consisted of nine conserved motifs that characterize DEAD-box RNA helicases and showed highly homology with DEAD-box RNA helicases of Oryza sativa, Arabidopsis thaliana and Pisum sativum. RT-PCR showed that expression level of ZmRH2 showed little difference in the leaf, root and ear between one pair of maize NIL (Near Isogenitic lines) of S-Mo17 Rf3Rf3 and S-Mo17 rf3rf3 , and the expression of ZmRH2 differed significantly in silk and pollen between S-Mo17 Rf3Rf3 and S-Mo17 rf3rf3 .

The differences at transcription level of the maize o2 mutation and genes related with the lysine formation and endosperm development were examined by cDNA microarray in two pairs of maize near-isogenic lines (NILs), Hz85(O2/O2) vs Hz85(o2/o2) and S7913(O2/O2) vs S7913 (o2/o2), respectively. Eighty-seven clones detected in both pairs of NILs were sequenced. Bioinformatics analyses showed that these clones represent 26 TUGs (tentative unique genes), 11 unnamed proteins and 6 new sequences. The TUGs are genes involved in several cell processes such as development, stress response, transportation, signal transduction, electron transfer chain, apoptosis, cell defense, metabolism, cell components and storage proteins. The molecular mechanisms of the formation of a soft, starchy, opaque mature endosperm in the o2 kernels were discussed based on the O2/o2 NILs.

The co-transcribed orf355-orf77 region of the mitochondrial genome is associated with S cytoplasmic male sterility (CMS-S) in maize; the amounts of its 1.6- and 2.8-kb transcripts were previously shown to be greatly reduced in fertility-restored microspores relative to the amounts in sterile plants. To investigate the mechanism underlying this reduction, detailed analysis of the 5' and 3' termini of these transcripts was conducted. Using 3' RACE analysis, the polyadenylation sites of the 1.6- and 2.8-kb transcripts were mapped adjacent to a 3' stem-loop, which may play an important role in stabilizing their 3' ends. No difference was found between the polyadenylation sites in sterile and fertility-restored microspores that could account for the differences in orf355-orf77 transcript levels. The 5' terminus of the 1.6-kb transcript was further studied by primer extension; the result revealed that there was a deletion of nine nucleotides only in fertility-restored microspores, and that this deletion eliminated a 5' stem-loop sequence. We propose that the elimination of the 5' stem-loop in the fertility-restored microspores could be the cause of the degradation of the 1.6-kb transcript. Because the 2.8-kb transcript can be cleaved to generate the 1.6-kb transcript, the amount of the 2.8-kb transcript is also reduced in fertility-restored microspores.

The normal gametophyte development of Stype cytoplasmic male sterility(CMS-S)of maize is disrupted,and then microspores are aborted which fail to pollination.A set of near isogenic lines of CMS,S-Mo17~(Rf3Rf3) and SMo17~(rf3rf3),was used,and TdT-mediated dUTP nick-end labeling(TUNEL),in situ immunolocalization of cytochrome C and DNA ladder assay were employed to confirm that programmed cell death(PCD) processes at cytology and DNA level.It is evident that the tapetal cells of fertile anthers kept intact until microspores,and then were disintegrated on later developmental stage.Whereas the tapetal cells of sertile anthers were earlier disintegrated when tetrad cells were disjoined.DNA ladder assay revealed that the cleavage of nuclear DNA into Oligonucleosome-sized fragments took place in pre-emergent anther,as well as in pollen cells from tasseled anther of S-Mo17~(rf3rf3).These results indicated that the abortion of sterile pollens was from not only prematuration of sterile anthers but also apoptosis of pollens.Results from TUNEL assay showed that nuclear DNA cleavage happened from microspore stage to the stage of pollen aborting in sterile anther cell,but not in fertile anther cell.These results also supported the conclusion based on DNA electrophoresis.In addition,the release of cytochrome C from mitochondria to cytoplasm is a pivotal signal of launching PCD.The analysis of immunolocalization of cytochrome C showed that cytochrome C was not released before microspores stage in S-(Rf3),but began to release when tetrad was disjoined in S-(rf3).During starch filling of pollen grain,DNA cleavage and the release of cytochrome C of tapetal cells were found in sterile and fertile anthers,however,these behaviors took place earlier in sterile anthr cell than in fertile anther cell.Tapetal cell of anther plays important functions during the development of microspores,such as providing nutrition,excreting β-1,3-glucanase to make microspores apart from tetrad.The release of pollen depends on the programmed death of anther cell too.The entire process of pollen development involves a series of fine modulation on molecular and cytological level.Each misregulation,such as abnormal excreting β-1,3-glucanase,prematuration of tapetal cells and abnormal trigger of PCD of anther cells,is likely to cause male sterility.The release of cytochrome C and the cleavage of DNA were observed at tetrad stage in sterile anthers but not in fertile anthers,implying that PCD which was launched earlier than pre-established programmer was a potential reason of pollen abortion in maize CMS-S.

Cytoplasmic male sterility (CMS) is thought to be due to an incompatibility of 2 genomes that results in pollen abortion. In CMS-S of maize (Zea mays L.), mitochondrialorf355-orf77 and the nuclear restorer of fertility interact to control fertility of gametophytes. Numerous studies demonstrated thatRf3 can regulate nuclear and mitochondrial gene expression and shows pleiotropic effects on the transcriptional level. Little is known, however, about the alteration of the global expression profile caused byRf3 substitution ofrf3 under S cytoplasm or about molecular fertility restoration mechanisms. In this study, cDNA microarray and suppression subtractive hybridization were used to reveal differentially expressed genes during pollen development by comparing a set ofRf3/rf3 near isogenic lines. A total of 137 tentative unique genes (TUGs) were identified at the transcriptional level. On the basis of functional category analysis, these TUGs were involved in a broad range of cellular and biochemical activities, including metabolism, cell structure, cell defense, and apoptosis, as well as signal transduction pathways. Northern blot analysis using 5 representative clones as probes confirmed differential expression among S-(Rf3) and S-(rf3). Especially in S-(Rf3), the expression patterns of genes associated with electron or H+ conduction and antiapoptosis genes (e.g., VADC2, BI-1, cystatin, 14-3-3) are distinctly different from in S-(rf3). Together with normalization of cellular and biochemical activities in S-(Rf3), we proposed thatRf3 might regulate accumulation of nuclear and mitochondrial gene transcripts directly or indirectly to inhibit multiple programmed cell death pathways in S-type cytoplasm allowing the normal developmental pathways to unfold.

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize ( Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.