Co-transfer of LEA and bZip Genes from Tamarix Confers Additive Salt and Osmotic Stress Tolerance in Transgenic Tobacco

In this study, the additive effects of a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene on salt and osmotic stress in Tamarix plants were analyzed. The constructs containing one or both of the LEA and bZIP genes were transformed into tobacco. Northern blot analysis showed the genes were overexpressed under the control of the CaMV 35S promoter in both dual and single gene-transgenic tobacco lines. The effects of salt and osmotic stress in transgenic tobacco plant were investigated. Following exposure to NaCl, mannitol, and PEG6000 stress, dual gene-transgenic lines showed higher seed generation and growth rates than single gene-transgenic lines and the wild-type. In response to NaCl stress, the dual gene-transgenic lines showed lower malondialdehyde and higher leaf chlorophyll content than single gene-transgenic lines and the wild-type. These results suggested that the co-expression of LEA and bZIP resulted in an additive enhancement of stress tolerance in dual gene-transgenic tobacco.     

Functional Characterisation of the Oil Palm Type 3 Metallothionein-like Gene (<Emphasis Type="Italic">MT3-B</Emphasis>) Promoter

In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns. Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3 homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues. The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with 80 μM of Zn²⁺ and Fe²⁺, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product.     

超重力胁迫下‘平邑甜茶’幼苗叶片生理指标及其转录组分析

该试验以‘平邑甜茶’60 d龄幼苗为材料,设置T₁(5 000 r/min×3 h)、T₂(5 000 r/min×6 h)、T₃(10 000 r/min×3 h)和T₄(10 000 r/min×6 h)超重力处理,以未进行超重力处理为对照(CK),考察超重力处理下幼苗叶片叶绿素、可溶性蛋白、丙二醛、脯氨酸含量和膜透性等与逆境相关的生理指标的变化,并对其进行转录组分析,初步探究‘平邑甜茶’在超重力处理下的转录组响应机制。结果表明：(1)超重力处理造成‘平邑甜茶’幼苗叶片由舒展状生长状态向不同萎缩生长状态转变,其中T₄处理萎缩生长状态明显。(2)超重力处理下,‘平邑甜茶’幼苗叶片叶绿素和丙二醛含量在T₃和T₄处理下显著高于CK, T₁和T₂处理下与CK无显著差异;叶片脯氨酸含量在各超重力处理下均显著高于CK;膜透性在T₄处理下显著高于CK, T₁、T₂、T₃处理与CK无显著差异;可溶性蛋白含量在各超重力处理下均不同程度降低,但均与CK差异不显著。(3)样品测序结果显示,大多数转录本的序列长度在1 000 nt以上,有37 725个,占全部转录本79.15%,且CK、T₂、T₄样品以及相对应的新基因中均以FPKM值小于5.0的转录本数为主,其转录本数依次分别有21 412、20 162、22 368个和1 352、1 411、1 406个,占比分别为45.78%、43.10%、47.82%和81.45%、85.00%、84.70%。(4)GO功能注释与富集分析显示,随着超重力处理强度的加大,差异表达基因在GO富集分类中存在明显不同的途径,说明‘平邑甜茶’对超重力的响应是复杂的,但主要集中在转录因子活性、蛋白质去磷酸化过程方面。(5)差异转录因子表达分析显示,随超重力处理强度的增加表达上调幅度比较大的基因家族有bZIP、WRKY和ERF,其中bZIP和WRKY转录因子家族中的基因表达量均随超重力处理强度的增加呈上升趋势,但ERF家族中的MD01G1177100、MD08G1096000、MD13G1130700基因表达量随超重力处理强度的增加有下降的趋势。研究表明,超重力处理对‘平邑甜茶’幼苗造成了不同程度的伤害,且叶片总叶绿素、丙二醛、脯氨酸含量以及膜透性均受到显著影响,故幼苗叶片中叶绿素、可溶性蛋白、丙二醛及脯氨酸含量以及细胞膜膜透性均可作为评价超重力胁迫的抗性指标;研究推测超重力胁迫对‘平邑甜茶’转录因子的影响主要集中在WRKY、bZIP和ERF转录因子家族。该研究结果为候选抗性基因的挖掘和筛选奠定了理论基础。