红肉苹果分裂素响应基因MdMYB308的克隆与表达分析

【目的】细胞分裂素是调控植物花青苷合成的重要激素,从新疆红肉苹果杂种一代优系‘紫红3号’中克隆得到分裂素响应基因Md MYB308,研究其在细胞分裂素调控苹果花青苷合成中的作用,为进一步完善红肉苹果育种的理论与技术体系提供参考。【方法】以红肉苹果‘紫红3号’(新疆红肉苹果与‘富士’杂交1代)的红色幼嫩叶片为外植体诱导的红色愈伤组织为试材,设计引物利用PCR克隆Md MYB308,对其进行生物信息学分析;并用不同浓度细胞分裂素处理,采用荧光定量PCR分析Md MYB308及花青苷合成相关基因的表达;通过酵母双杂交试验、荧光双分子互补试验验证Md MYB308与Mdb HLH3的互作关系。【结果】在‘紫红3号’中克隆获得Md MYB308全长,其包含768 bp完整的开放阅读框,编码255个氨基酸,预测其编码蛋白质分子量为28.37 kD,等电点为8.94;系统进化树分析表明,Md MYB308与At MYB4、Fa MYB1、At MYBL2在同一个进化枝上,氨基酸序列比对发现,Md MYB308蛋白存在EAR抑制序列;提高6-BA浓度有利于苹果愈伤组织花青苷的累积,与无细胞分裂素处理相比,1 mg·L~(-1) 6-BA处理愈伤花青苷合成结构基因Md CHS、Md DFR、Md UFGT与转录基因Md MYB10、Mdb HLH3的表达量升高,而Md MYB308表达被抑制;酵母双杂交与荧光双分子互补试验表明,Md MYB308与Mdb HLH3能相互作用。【结论】细胞分裂素(6-BA)可能通过抑制Md MYB308的表达影响Md MYB308与Mdb HLH3的结合从而促进花青苷的累积。     

11个苹果野生砧木品种低温处理抗性指标的综合评价

对苹果野生砧木品种进行抗寒性综合评价,为耐寒性砧木的开发利用提供科学依据。以11个野生砧木品种为试验材料,在-10~-45℃设置7个低温梯度处理,对枝条萌芽率,电导率、脯氨酸、丙二醛含量进行研究。结果表明,低温处理后,围矮4号和Sdw1萌芽率迅速下降,11个野生砧木品种的相对电导率随处理温度下降呈“S”形增加,计算得半致死温度（LT₅₀）介于-22.32~-38.58℃。丙二醛含量呈双峰变化趋势,Sdw1、串铃海棠、围矮4号丙二醛含量较高,围矮2号、Spy227、特大果山定子丙二醛含量低且稳定。围矮4号、Sdw1、串铃海棠Pro累积较早,围矮2号和特大果山定子脯氨酸含量最大。利用隶属函数法进行抗寒性综合评价,11个野生砧木抗寒性强弱顺序为围矮2号＞特大果山定子＞Spy227＞八棱海棠＞围矮3号＞克勒沟林场大果山定子＞围矮1号＞五峰山4号＞串铃海棠＞围矮4号＞Sdw1,与根据LT₅₀所得抗寒性排序相近。     

苹果生长素响应因子MdARF5的克隆与功能鉴定

【目的】分离苹果生长素响应因子MdARF5（Auxin Response Factor 5）,分析其对生长素的响应,鉴定其在调节花青苷合成过程中的作用,揭示MdARF5的生物学功能,为进一步研究生长素对花青苷的调节提供理论依据。【方法】以‘嘎拉’苹果（Malus×domestica ‘Royal Gala’）为材料,利用同源克隆技术,克隆得到一个ARF（Auxin Response Factor）转录因子,并将其命名为MdARF5。利用MEGA5.0软件构建多物种间系统进化树。通过农杆菌介导的遗传转化获得转基因苹果愈伤组织。比较野生型和转基因苹果愈伤组织花青苷积累的差异。利用烟草叶片瞬时转化试验,分析MdARF5对MdMYB1的转录调控。【结果】克隆获得苹果生长素响应因子MdARF5（序列号：MDP0000143749）,该基因CDS为2 691 bp,编码含有896个氨基酸的蛋白。系统进化树分析表明,苹果MdARF5与梨PbARF5同源性最高。基因表达分析显示,该基因响应生长素处理,并且与花青苷合成相关基因表现出相反的表达模式。在苹果愈伤组织中超表达MdARF5,其花青苷积累较野生型显著降低,表明MdARF5在调控花青苷积累过程中发挥重要作用。对苹果MdMYB1启动子序列进行分析,发现其序列包含一个MdARF5的结合位点。烟草瞬时表达试验显示,MdARF5能够抑制MdMYB1的表达。【结论】推测苹果MdARF5可能通过直接抑制MdMYB1的表达负调节花青苷的积累。     

Physiological mechanisms of resistance to cold stress associated with 10 elite apple rootstocks

A study was conducted in attempting to identify the cold-resistant apple rootstocks and to establish a comprehensive evaluation system. In this study, 10 elite apple dwarfing rootstocks(GM256, JM7, M26, M7, SC1, SH1, SH38, SH6, M9, and T337) were employed for the experiment and the following parameters were investigated under different low temperature stress conditions(0, –15, –20, –25, –30, and –35°C): the changes of the relative electrical conductivity(REC), anthocyanin content, protein content, soluble sugar content, soluble starch content, proline content, malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, and peroxidase(POD) activity of the dormant branches. The inflection temperature that could represent the plant tissue semi-lethal temperature(LT_(50)) was obtained by the measurements of REC. The LT_(50) was used to evaluate eight other indices. The results showed that there was no significant correlation between LT_(50) and POD activity as well as between the soluble sugar, protein and proline contents at 0 and –15°C. Soluble starch content at 0 and –15°C and anthocyanin content at –15–(–30)°C were significantly but negatively correlated to the LT50 and the MDA content at 0–(–20)°C was significantly positively correlated to the LT_(50). Statistical analysis based on principal component analysis and LT50 showed that cold resistant apple rootstocks in the decreasing order from high to low as GM256, SH6, SH38, SH1, SC1, M26, M7, JM7, T337, and M9.     

苹果矮化砧木抗寒性

【目的】研究苹果矮化砧木抗寒性,为选育、鉴定和推广应用抗寒的苹果矮化砧木提供理论依据。【方法】以62-396、BP-176、Y-1、KM23 4种苹果矮化砧木1年生枝条为试材,进行不同低温处理,采用电导法配合Logistic方程计算不同参试休眠枝条的半致死温度(LT₅₀),并结合冻害分级、恢复生长试验和田间冻害调查,研究苹果矮化砧木的抗寒性。【结果】在低温胁迫下,枝条的电解质渗出率与相应的低温呈极显著负相关;62-396、BP-176、Y-1、KM23的半致死温度分别为-42.56、-37.52、-43.79和-40.77℃;低温处理后各枝条的萌芽率高低依次为Y-1＞62-396＞KM23＞BP-176;田间调查结果显示,BP-176抗寒性最差。【结论】4种苹果矮化砧木的抗寒性强弱依次为Y-1＞62-396＞KM23＞BP-176。苹果矮化砧木Y-1和62-396具有较大的应用潜力,可以作为苹果抗寒矮化砧木在伊犁河谷进行生产应用。     

苹果WRKY基因家族生物信息学及表达分析

【目的】鉴定苹果（Malus domesticaBorkh.）基因组上132个WRKY基因,为研究苹果WRKY转录因子在非生物和生物胁迫以及生长和发育过程中的调控作用奠定相关理论基础,也为进一步分析苹果WRKY基因提供信息。【方法】利用HMMER 3.0软件,通过WRKY保守域全蛋白序列PF03106用于鉴定苹果WRKY基因。采用WebLogo 3、DNAMAN 5.0、MapInspect、MEME和MEGA5.1等软件对其蛋白序列进行生物信息学分析。采用RT-PCR技术检测苹果WRKY基因的组织表达情况。【结果】鉴定得到132个苹果WRKY基因。分组鉴定和进化树分析结果显示,苹果WRKY蛋白分为I、II和III类型,I组共有24个成员可进一步分为I-C和I-N亚组,其锌指结构是C₂H₂类型（CX₄CX_22-23HXH）。II组含有1个WRKY区域共有79个成员,可进一步分为II-a、II-b、II-c、II-d和II-e亚组,分别有8、12、31、14和14个成员,其锌指结构为C₂H₂类型（CX_4-5CX₂₃HXH）。III组共有29个成员,其锌指结构为C₂HC类型（CX₇CX_23-24HXC）;WRKY结构域分析显示,其高度保守,绝大多数都含有WRKYGQK七肽和锌指结构;染色体定位分析显示,苹果WRKY分布于苹果17条染色体中,呈不均匀分布。染色体1和9上分布最多,为13个;其次是染色体12,分布12个;染色体2、5和14分布最少,为4个;基因结构分析表明,MdWRKY基因家族多数由2-5个外显子组成,基因结构进化高度保守;保守元件分析表明,MdWRKY基因家族包含10个保守元件：元件1-6为WRKY盒;元件7-10为未知盒。MdWRKY基因家族都包含有WRKY盒,I组中含有2个WRKY盒,II-a和II-b亚组中含有未知元件8,III组中含有未知元件7和9。半定量结果显示,12个MdWRKY均在根、茎、叶、花和果中表达,且呈现出多种相对表达模式。【结论】苹果WRKY基因家族结构高度保守,可能参与调控苹果生长和发育等过程。     

Overexpression of AmDUF1517 enhanced tolerance to salinity,drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmDUF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmDUF1517 cotton displayed significantly higher antioxidant enzyme (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione S-transferase (GST)) activity and less reactive oxygen species (ROS) accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.     

四种苹果砧木幼苗对NaCl胁迫的生理响应、根系生长及耐盐性评价

【目的】探讨盐胁迫下四种苹果砧木幼苗的根系构型和生理响应,评价其耐盐性能.【方法】以6叶龄八棱海棠(BL)、垂丝海棠(CS)、花叶海棠(HY)、新疆野苹果(XJ)幼苗为试材,采用Hoagland营养液水培的方式,在营养液中加2.0mg/g NaCl模拟盐胁迫.【结果】低盐胁迫下,四种苹果砧木幼苗根系的根长、根尖数、根表面积和根系活力缓慢下降;叶绿素含量呈现先升后降的趋势.随胁迫时间的延长,丙二醛(MDA)含量胁迫前期缓慢上升,胁迫后期急剧下降.过氧化物歧化酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性随胁迫时间延长均呈先升后降的趋势,有明显峰值.【结论】根据隶属函数总和的大小对苹果砧木幼苗的耐盐性能进行排序,四种砧木耐盐性由强到弱的顺序为:XJCSHYBL.     

利用苹果矮化砧木进行密植丰产栽培的意见

 本文总结了中国30多年来对苹果矮化砧木的利用工作，依据大范围的区域试验结果，论述了矮化砧木在苹果密植丰产栽培中的作用，提出了利用苹果矮砧的区划意见，以及与矮砧苹果相适应的系列栽培技术，最后就矮化砧木利用中的看法进行了讨论。作者认为在中国的主要苹果产区如渤海湾、胶东、黄河故道、秦岭北麓以及渭北高原等地均可栽培矮砧苹果，现在已经有了比较成熟的经验，因地制宜地选择利用矮化砧木，将对提高中国的苹果栽培水平起到积极作用。     

Comprehensive evaluation of tolerance to alkali stress by 17 genotypes of apple rootstocks

Alkaline soils have a great influence on apple production in Northern China. Therefore, comprehensive evaluations of tolerance to such stress are important when selecting the most suitable apple rootstocks. We used hydroponics culturing to test 17 genotypes of apple rootstocks after treatment with 1:1 Na2CO3 and Na HCO3. When compared with the normally grown controls, stressed plants produced fewer new leaves, and had shorter roots and shoots and lower fresh and dry weights after 15 d of exposure to alkaline conditions. Their root/shoot ratios were also reduced, indicating that the roots had been severely damaged. For all stressed rootstocks, electrolyte leakage(EL) and the concentration of malondialdehyde(MDA) increased while levels of chlorophyll decreased. Changes in root activity(up or down), as well as the activities of peroxidase(POD), superoxide dismutase(SOD), and catalase(CAT) were rootstock-dependent, possibly reflecting their differences in alkali tolerance. Using alkali injury index(AI), adversity resistance coefficients(ARC), cluster analysis, and evaluation of their physiological responses, we classified these 17 genotypes into three groups:(1) high tolerance: Hubeihaitang, Wushanbianyehaitang, Laoshanhaitang Ls2, Xiaojinbianyehaitang, and Fupingqiuzi;(2) moderate tolerance: Pingyitiancha, Laoshanhaitang Ls3, Hubeihaitang A1, Deqinhaitang, Balenghaitang, Maoshandingzi, Shandingzi, and Xinjiangyepingguo; or(3) low tolerance: Pingdinghaitang, Hongsanyehaitang, Xiaojinhaitang, and Sanyehaitang. These results will significantly contribute to the selection of the most suitable materials for rootstocks with desired levels of tolerance to alkali stress.     

THE ANTHOCYANIN BIOSYNTHETIC REGULATOR MDMYB1 POSITIVELY REGULATES ASCORBIC ACID BIOSYNTHESIS IN APPLE

• The contents of anthocyanin and AsA in red-flesh apples are higher than that in non-red-flesh apples. • The anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. • MdMYB1-MdDHAR module may play a key role in AsA-DHA homeostasis. Ascorbic acid (AsA, vitamin C) is involved in the regulation of many aspects of plant growth and development. It is an essential micronutrient for humans and can prevent scurvy, maintain the health of gums and blood vessels, reduce the level of plasma cholesterol and enhance the immune systen. Apple cultivars Orin and Guanghui were crossed to obtain a group of hybrid offspring with and without red flesh in the course of assessing apple germplasm resources. Unexpectedly, the red-flesh apples had higher AsA contents than other apples. Further studies showed that the anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. This finding reveals the mechanism leading to high AsA levels in red-flesh apples and suggests a new idea to cultivate red-flesh apples with high AsA contents and produce AsA efficiently and without pollution.     

不同苹果砧木实生苗对盐碱复合胁迫的生理响应

【目的】综合评价不同苹果砧木的耐盐碱性,筛选适合新疆南疆盐碱土壤栽培的苹果砧木。【方法】以湖北海棠、小金海棠、八棱海棠、平邑甜茶、野苹果、白海棠、山定子7个4年生苹果砧木为材料,将NaCl、Na₂SO₄、NaHCO₃、Na₂CO₃按1∶9∶9∶1的比例混合,采用盆栽浇灌1/2 Hoagland营养液,模拟浓度为0、15、30、45、60 mmol/L的盐碱复合胁迫,测定7个苹果砧木在盐碱复合胁迫下叶片的叶绿素含量、相对电导率、脯氨酸含量等5项生理指标,利用隶属函数法综合评价各砧木的耐盐性。【结果】随着胁迫强度的增大,各砧木叶片叶绿素含量不断减小,相对电导率和脯氨酸含量总体呈上升趋势,可溶性总糖含量总体呈先上升后下降,可溶性蛋白含量呈先上升后下降和“升高—降低—升高”2种变化趋势。【结论】7种供试苹果砧木的耐盐碱能力由强到弱依次为八棱海棠>野苹果>平邑甜茶>小金海棠>山定子>白海棠>湖北海棠。     

小金海棠×M9后代杂种鉴定及倍性分离分析

为选育矮化和耐缺铁的苹果砧木,配置了小金海棠×M9杂交组合,首先筛选M9纯合显性SSR分子标记并利用该标记鉴定小金海棠×M9的有性杂种后代,之后利用流式细胞术分析杂交后代中的倍性分离。结果表明:SSR标记CH02d12-160可作为鉴定杂种后代的特异标记。在1 285株小金海棠×M9实生苗中,有284株为有性杂种后代,其余1 001株为小金海棠无融合生殖后代,无融合生殖率为77.90%。在284株小金海棠×M9有性杂种后代中,138株的样品得到可靠倍性鉴定结果,其中2x、3x、4x和5x分别为5(3.62%)、21(15.22%)、63(45.65%)和49株(35.51%)。     

A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG). This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×10⁴ to 1.4×10⁸ cells μg^–1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×10⁸ CLas cells μg^–1 DNA were 30 and 40% at 30 and 40 DAG, respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro. This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.     

Study on Mutant Induction of Gladiolus by in vitro Culture of Petals

Petals of Gladiolus 'Rose Supreme' were used to establish regeneration system. Developmental characteristics of petals were observed. A total of 94 plantlets of petal somaclonal line were marked in Mj ...     

我国苹果产业70年发展历程与展望

目的研究干旱胁迫对苹果不同中间砧及其上嫁接品种的叶片光合特性及抗氧化酶活性的影响。方法以河北农业大学自主选育的苹果矮化砧木冀砧3号及优系15-1、1-8、14-7、10-1和22-46为试验材料,研究干旱胁迫下苹果砧木及其上嫁接天红2号的叶片光合参数和抗氧化酶活性的变化,并利用隶属函数法分析不同中间砧对干旱胁迫的响应差异。结果干旱胁迫下,不同苹果砧木及砧穗组合的光合作用受到不同程度的影响;叶片初始荧光与可变荧光和最大荧光比值与对照组相比差异显著;随着干旱胁迫时间的延长,砧木叶片抗氧化物酶(SOD、POD和CAT)活性呈现先升高后降低的趋势,嫁接品种叶片的SOD和CAT活性整体呈升高的趋势;砧木14-7及其上嫁接天红2号叶片的光合作用和3种抗氧化酶活性对干旱胁迫响应相对较弱,而砧木22-46和其上嫁接品种响应较强。隶属函数分析表明：6种砧木抗旱性为14-7>15-1>冀砧3号>10-1>1-8>22-46。结论干旱胁迫下,苹果6种不同中间砧及其上嫁接品种的光合特性和抗氧化酶活性存在差异。