木薯(Manihot esculenta)AKT1基因的克隆及生物信息学分析

植物细胞中的K^+通道蛋白AKT1(Arabidopsis K^+transpoter 1)主要负责吸收K^+。本研究基于木薯基因组数据库信息,利用PCR技术从木薯中克隆一个MeAKT1基因,该基因全长2634 bp,编码877个氨基酸,生物信息学分析表明MeAKT1含有10个外显子和9个内含子,编码的氨基酸分子量为99.02 kD,等电点为8.43,属于稳定蛋白。MeAKT1包含有5个跨膜区域,N端含有1个Ion_trans结构域,C端有1个KHA结构域,其二级结构以α-螺旋和无规则卷曲为主,进化树分析发现MeAKT1与蓖麻RcAKT1的亲缘关系最近。通过对MeAKT1蛋白的生物信息学分析有助于下一步深入研究MeAKT1在木薯耐贫瘠过程中的功能。     

毛薯的营养成分分析和聚类分析

为选育出优良的毛薯种质,本研究以海南大学薯蓣种质资源圃里的80份毛薯为材料,以河南焦作温县的垆土铁棍山药为对照,采用苯酚硫酸法、考马斯亮蓝法以及蒽酮比色法对其干物质、粗多糖、蛋白、可溶性糖和淀粉含量进行测定和比较分析。结果表明:毛薯的干物质含量介于18.62%~38.88%,粗多糖含量介于2.88%~15.82%、蛋白含量介于5.14%~10.71%、可溶性糖含量介于0.1%~13.84%、淀粉含量介于11.94%~64.28%。与对照山药相比,Ds96所测品质均优于对照,可优先选择推广。此外,毛薯各营养品质之间存在着一定的相关性,干物质与糖类存在显著负相关,与蛋白和淀粉表现为正相关。通过对80份毛薯的营养品质进行聚类分析,在欧式距离为3.17时,可将其分为八类,按类群来看,类群Ⅳ各营养价值适中,其他类群在某些营养上比较优秀,可根据需要进行优良资源选择。      

不同营养条件对羊肚菌菌核形成的影响

菌核形成与羊肚菌(Morchella esculenta)子实体的形成有密切的关系。以PDA培养基为基础培养基,通过添加其他物质培养羊肚菌菌丝,观察菌核生长情况,从而初步判断其形成菌核的培养基限定条件。结果表明,碳源营养添加物为淀粉和乳糖,浓度为10 g·L^-1;氮源添加物为NH4HCO3、KNO3和甘氨酸,浓度为250 mg·L^-1;添加KH2PO4,浓度为1 g·L^-1,均能较好地促进羊肚菌菌核的形成。     

木薯有性四倍体遗传稳定性分析

木薯作为中国重要的经济作物,其优良品种的选育一直是木薯科研工作的重点,而多倍体的巨型性特点为其育种提供了新的思路,目前多倍体研究大多是体细胞加倍,有性多倍化研究起步较晚,而本课题组通过2n配子有性活体杂交,成功获得了首例木薯有性四倍体植株。为了研究木薯有性四倍体的遗传稳定性,本研究分别采用:田间的农艺性状观测(生长稳定期)、流式细胞术、叶片染色体计数、蛋白质含量测定/蛋白质聚丙烯酰胺凝胶电泳以及淀粉含量测定等方法进行鉴定。结果显示:木薯有性四倍体各株系农艺性状均保持了母株器官巨型性的多倍体特征;流式细胞仪分析显示,木薯有性四倍体各株系叶片DNA含量均为二倍体亲本(SC5)的两倍,相对应的叶片染色体数目也为二倍体亲本(SC5)的两倍,且视野中四倍体细胞所占比例均在90%以上;各株系蛋白质含量均小于二倍体对照组,且有性多倍体株系电泳谱带基本相似;在淀粉含量测定中,其各株系淀粉含量均小于对照组,且各株系之间含量波动较小。本研究结果表明木薯有性四倍体植株具有较好的遗传稳定性,对木薯多倍体品种的选育具有重要意义。     

木薯金属硫蛋白基因的克隆和表达分析

金属硫蛋白(metallothionein,MT)分子量小,半胱氨酸含量高,具有消除离子自由基,减弱重金属毒性和减少体内活性氧积累等功能。为探究木薯金属硫蛋白基因(MeMT)的原核表达能力和对活性氧的耐受性,本研究通过木薯cDNA克隆获取MeMT并将其连接至pET-28a,在BL21(DE3)菌株中成功诱导其表达。H2O2耐受性分析发现,与空载菌株相比,表达MeMT蛋白的DE3菌株对H2O2的耐受水平较高。RT-qPCR技术检测显示,在H2O2胁迫下,3 h时木薯叶片中MeMT的表达水平明显升高,但6 h时恢复甚至略低于对照组,证明MeMT在木薯中参与ROS应答过程。该结果对研究木薯抗逆性和产量的提高具有重要意义。     

基于表型性状与简单重复序列标记的浙江省芋种质资源遗传多样性比较

为探讨浙江省地方芋种质资源的遗传多样性,并挖掘特异芋种质,对25份浙江省地方芋种质资源进行调查鉴定,对15个表型性状进行了遗传多样性分析、主成分分析、表型性状与简单重复序列(SSR)标记相结合的聚类分析。结果表明,浙江省芋种质遗传多样性丰富,变异系数与多样性指数均值分别为31.32%与0.97。主成分分析显示,前5个主成分累积特征值为10.871,累计贡献率为77.649%,主成分1反映了多子芋株型综合因子,主成分2反映了芋叶形状综合因子,主成分3反映了母芋形状综合因子,主成分4、5分别代表母芋表皮棕毛、母芋颜色因子;另外,孙芋形状、母芋芽色、叶心色斑颜色、叶柄中下部颜色、母芋表皮棕毛可作为芋种质鉴别优先观测的形态性状指标。基于表型性状的聚类在遗传距离4.171处可将所有种质分为5个类别,其中第Ⅲ类与第Ⅴ类的子孙芋形状均为卵圆,商品性好。此外,在20份多子芋中还挖掘出2份紫红柄特异芋种质。20对SSR引物在所有资源中扩增共获得53个条带,具有多态性的条带为48条,多态性比率为90.6%,多态信息含量(PIC值)为0.22~0.64;SSR分子标记聚类在DICE遗传相似系数0.721处将所有种质分为5个类别,与表型性状聚类结果有类似之处,但又有所不同。2种不同的聚类方式在芋种质资源的鉴别中具有互补作用,均表明了亲缘关系的远近与地理来源并无直接关系。上述结果将为芋种质资源进一步保护、利用与创新提供重要的理论基础。     

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

Successful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.     

普通羊肚菌密码子偏好性分析

采用CodonW1.4.2软件和CUSP程序,以普通羊肚菌(Morchella conica)全基因组蛋白质编码序列(coding sequence,CDS)为对象,解析了该菌的有效密码子数(effective number of codon,ENC)、密码子3个位点的 GC含量、相对同义密码子使用度(relative synonymous codon usage,RSCU)和高表达优越密码子。结果表明：普通羊肚菌全基因组密码子第2位密码子的 GC含量明显低于第1位和第3位,第3位密码子与第1位含量差异不大,分别为57.8%和56.8%,RSCU 值大于等于1的密码子总共35个,其中以 G 或 C 结尾的25个,占71.4%,确定了25个高表达优越密码子。     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.