水稻( Oryza sativa? L.)是亚洲主要粮食作物.我国是水稻原产地之一,种植历史悠久.武陵山区包括湖南、湖北、重庆、贵州、四川相邻的一些山地.该地是我国扶贫攻坚的重点地区,分布有一定数量的富硒地区,发展水稻富硒种植对于发展当地经济有着积极意义.硒是人体必须的营养元素,它具有增强人体免疫力、防癌抗癌、提高机体抗氧化等多种功能,对人体健康起着重要作用.由于土壤中的硒含量不均匀,仅靠水稻吸收土壤中的硒元素难以达到富硒标准,通过对叶片喷施硒源,使其在水稻植株体内转化为有机硒便成了一个应用范围较广的技术.达到富硒标准的稻谷用途更广,更受市场欢迎,比普通水稻增值20％～50％,发展前景可观.水稻富硒种植中应掌握以下技术环节.

在“互联网+”的大背景下，翻转课堂教学、过程考核和个性化教学思想融入植物生理学实验教学全过程，将微课、微视频和在线测评通过网络平台应用于该门课程的教学，建立“习、问、讲、演、做、导、结”七步标准化教学体系，在实验教学的各个环节，针对性的培养学生的学习力、思考力、动手力和沟通力，取得了良好的教学效果。

Transgenic citrange lines with rolABC genes behave rosette branching and extreme dwarfing. To explore the regulatory mechanism of plant hormones in axillary shoot growth of transgenic citrange, a full length cDNA of CPMAX2 was cloned from citrange [Citrus sinensis (L.) Osb × Poncirus trifoliate (L.) Raf.] by RT-PCR in this study. The expression of CPMAX2 was detected in axillary tender leaves of 3 transgenic citrange lines and the wild type. Additionally, we constructed an over-expression vector CPMAX2-pCAMBIA1301 for further study. The results showed that the cDNA sequence and its putative peptide sequence shared 99.66% and 99.14% of identity with its Citrus sinensis ortholog MAX2. The deduced amino acid sequence contained putatively one F-box and two LRR repeat domains that are highly conserved in MAX2 genes. CPMAX2 was obviously down-expressed in tender leaves of 3 transgenic citrange lines compared with the wild type. The results suggest CPMAX2 play an important role in regulating the rosette shoot growth in transgenic citrange with rolABC genes.

转rolABC基因枳橙莲座状分枝、矮化性状突出.为探索其侧枝生长的植物激素调控机制,通过RT-PCR法从枳橙中克隆到独脚金内酯信号转导关键元件CPMAX2基因,分析了该基因在转rolABC基因枳橙腋生嫩叶中的转录表达,构建了CPMAX2植物过表达载体.研究表明:CPMAX2与甜橙中同源基因一致性为99.66％,编码694个氨基酸,具有一个典型的F-box蛋白结构域和两个LRR重复区,与甜橙MAX2氨基酸序列一致性为99.14％.CPMAX在转rolABC基因枳橙3个株系腋生嫩叶中的转录表达均比野生型显著下调.试验表明CPMAX2在转rolABC基因枳橙莲座型分枝生长中扮演重要角色.

Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.

The transcription factors TERF1 gene was transferred into 'Succari' sweet orange [Citrus sinensis (L.) Osbeck] via Agrobacterium infection and 7 transgenic clones were obtained. Three transgenic clones were further tested for tolerance to water deficiency, low temperature and their resistance to citrus canker disease (Xanthomonas axonopodis pv. citri). Under 10% of soil relative water content (SRWC), transgenic clones showed stronger tolerance than the wild type, with normal growing, leas leaf chlorophyll content decrease, higher Proline (Pro) accumulation and activity of the protective enzymes (CAT, POD and SOD). Simultaneously, transgenic clones showed stronger tolerance to low temperature stress. When plants being treated at -10 degrees C for 30 min, all the wild type plants completely died within 5 days, while the transgenic plants merely revealed severe damages on the shoots and leaves, the main stem and the roots were still alive. The terf1 transgenic clones displayed resistance to citrus canker disease. In the in vitro assay and vivo assay, transgenic clones obviously decreased the disease incidence rate. When the transgenic clones were inoculated with 104 and 105 cfu/ml of citrus bacterial canker pathogen, no symptom occurred. The results indicated that the tested transgenic clones showed obvious tolerance to abiotic stresses and resistance to canker disease. Further investigation in field is going on.

The potted seedlings of transgenic citrange with rol ABC genes were used as trial materials, the sensitive response to gibberellins(GAs) was tested by spraying GA_3 regularly.The expressions of GA20ox1 and rol A,B and C genes were detected by quantitative reverse transcription PCR and the phytohormone content of tender bud was quantified by GC/MS method.The results demonstrated that the transgenic citrange was neither defective genotype of GA_3,nor unsensitive genotype of GA_3.The contents of IAA(P0.01),GA_1 and GA_4 decreased(P0.05) while the peroxidase activity increased markedly in the tender bud of transgenic citrange(P0.01).The GA20ox1 expression quantity markedly decreased in apical bud(P0.01).The expression of rol C gene and GA20ox1 gene reveal negative correlation in tender bud and tender stems.The rol genes' expression could inhibit synthesis of GAs by down regulating the expression of GA20ox1 gene in bud and lower bioactive GAs could suppress citrange growth,then it played an important role in constructing dwarfing characteristics of transgenic citrange with rol ABC genes.

45 days old leaves from transformed sweet orange with pthA-nls gene,different organs(roots,shoots,leaves and buds) from Citrange,and 45 days old leaves from untransformed Trifoliate,Satsuma Mandarin,Citron,Pomelo,Bingtang sweet orange,Citrange were tested.The stability of expression ofβ-actin gene was identified by SYBR GreenⅠfluorescent dye method.The results demonstrated the expression ofβ-actin gene in different varieties and tissues of citrus in a constant states.β-actin gene can be used as reference gene for quantitative PCR analysis in citrus and the relative expression level of pthA-nls gene in different transgenic sweet orange was detected withβ-actin as reference gene.

B,D,E transgenic clones with rolABC genes and wildtype plants were used as materials to construct binary standard curves of rol genes and β-actin gene with SYBR Green I fluorescent dye.After normalizing the influence of amplification efficiency with different primers in qRT-PCR,comparative Delta-delta Ct method was applied to develop suitable qRT-PCR method for analysising the expression of rolA,rolB,rolC genes in 5 tested tissues of B,D,E clones.The primary result showed that rolC expressed highest among 3 rol genes in tender stem,tender leaf,functional leaf and bark,rolA gene's expression in the middle level and mainly in tender stem,and rolB expressed lowest and mainly in root.

The appearance of variedness and biological characteristics of three variegated sport varieties and their mother plant of Loropetalum chinense var. rubrum ‘Mizhi Meihong’were compared with virological identification,anatomy of blade,content of pigments and photosynthetic traits. The leaf extracts from the plants were used to inoculate Glycine max, Cucumis sativus,Capsicum annuum,Solanum lycopersicum,L. chinense var. rubrum. The results showed that the inoculated plants grew well,and did not exhibit any virus symptom. No viral granule was detected by TEM. The bud sport varieties were able to maintain their trait of variegated color by cutting propagation,and they had higher resistance to plant diseases and insect pests and were suitable to growing in field in Hunan Province. There was remarkable difference in thickness of leaves,upper epidermis and lower epidermis among 4 plant specimens. The contents of carotenoid and anthocyanin of variegated sport leaves were distinct dissimilar among 3 sport varieties,and the net photosynthetic rates were higher than their mother plants. The sport varieties had significant distinct flower colors as well as leaf colors from their mother plant. The varieties were elite germ plasm resource for breeding of L. chinense var. rubrum. Especially,the No. 2 sport variety could be a prospective commercial product.

Citranges transferred with phyB gene were detected at the molecular level and their biological characteristics were analyzed.PCR,RT-PCT and PCR-Southern blot analysis indicated that phyB gene was successfully integrated into citrange genome and was transcribed in citrange.Compared with the non-transgenic plant,the transgenic plant was dwarf,with larger shoot insertion angle and smaller leaf area,and was higher in chlorophyll content,net photosynthesis rate,light saturation point and specific leaf weight.Determination of endogenous hormone by HPLC indicated that the content of GA1,GA4,ABA and Z of the transgenic plant was 32.7%,50%,121% and 17.8% higher than that of the non-transgenic plant,respectively,whereas the content of IAA was lower than that of the non-transgenic plant.(GA1+ GA4)/ABA and Z/ABA ratios of the transgenic plant were 38.4% and 43.2% lower than those of the control,respectively.

To study Ptcor8 expression related to the defoliation of Poncirus trifoliate Raf. and the temperature, 2-year-old seedlings of 60 potted Poncirus trifoliata Raf. with similar diameter, in the temperature-controlled chamber (20～25 ℃), were transferred to the field every 10 d from the 14th November, 2008 to January of next year, meanwhile, another 10 seedlings were put in the field as the control. The Poncirus trifoliate Raf. seedlings in the temperature-controlled chamber and those transferred to the field after the 14th December were non-deciduous. However, those transferred to the field before the 14th December was deciduous, and their defoliating period delayed along with the time outside the temperature-controlled chamber. In the deciduous and the non-deciduous the expression of Ptcor8 gene was both detected. Furthermore, by qPCR, the more changes of temperature and the longer duration of low temperature, the higher expression of Ptcor8. Therefore, the expression of Ptcor8 gene may not be directly related to the physiological defoliation of Poncirus trifoliate Raf., but rather closely related to low temperature induction.

A parthenocarpy gene (DefH9-iaaM) was introduced into Succari to obtain possible seedless transformants, by using internodal stem segments as explants to establish an efficient regeneration and transgenic system. The results indicated that 30-day-old seedlings were the most efficient explants. The co-culture in darkness for 3～6 days gave a rise to good regeneration. The optimum medium for adventitious buds regeneration was MS+6-BA 5.0 mg/L. And MS medium supplied with 6-BA1.0 mg/L and NAA0.1 mg/L showed the highest bud multiplication rate,3.3 buds on each internodal segments. The shoots successfully rooted after 20 days in 0.5 mg/L NAA. After Agrobacterium-mediated transformation,6 transgenic plants out of 17 regenerants were identified by PCR.

The phyB gene was transferred into the genome of Poncirus trifoliate in vitro with explants of epicotyl mediated by Agrobacterium.Total 27 strains with plant resistance plants were regenerated.The presence of specific targeted strips of phyB gene in the 5 regenerated plants was confirmed by PCR analysis.RT-PCR analysis indicated the regular expression of transgene phyB in two transgenic clones,phyB3 and phyB4 in omparison with the control plants,the phyB transgenic plants had higher net photosynthesis rate.All the regenerated transgenic plants,as compared with the untransformed clones,showed lower height,shorter interval between internodes and smaller leaf area.