Deping Xu

Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.

Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests.

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.

Systemic induction of a potato pin2 promoter by wounding, methyl iasmonate, and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial β-glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5′-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5′ deletions fused with the bacterial β-glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.

Analysis of rice genes in transgenic plants.

Publisher Summary This chapter discusses the information on molecular analyses of rice genes by in vivo and in vitro methods and in transgenic plants. It selected examples of several important types of rice genes, particularly those about which ones have first-hand information. Another consideration in choosing specific genes for discussion involves selection of those genes that are unique to plants: for example, genes related to seed storage proteins in rice and other monocotyledonous plants, such as wheat and barley. The response of rice phytochrome genes to light is another such example of gene regulation unique to plants. However, the development of specific applications, such as attempts to produce insectresistant transgenic rice plants, is a unique and important application of plant biotechnology. Moreover, in plant research, work on dicotyledonous plants such as tobacco and Arabidopsis is more advanced than that on monocotyledonous plants, such as rice and wheat because these dicots are easier to transform and regenerate more plants than monocots.

Development of Promoter Systems for the Expression of Foreign Genes in Transgenic Cereals

Recent advances in monocot transformation technology have resulted in the routine production of transgenic plants for an increasing number of cereal species. With a view towards the improvement of cereal quality by genetic engineering attention is beginning to focus on the identification and utilisation of promoters that will be used to control the expression of agronomically important traits in transformed cereals.

Production and Analysis of Transgenic Rice Plants

Several gene constructs were used to transform rice protoplasts and several hundred transgenic plants were regenerated. in one construct, the 5’ region of the rice actin 1 gene (Act1) was ligated to a bacterial β-glucuronidase (GUS) gene. The Act1/Gus construct was expressed constitutively in transgenic plants because GUS activity was found in all tissues tested. Quantitative analysis showed that in two lines of transgenic plants, the level of GUS protein represents 3% and 0.5% of total soluble protein, respectively. In two other constructs, the 5’ region of the potato protease inhibitor lI (PlNII) gene was joined to the Gus gene to produce: pin2/Gus and pin2/Act1 intron/Gus. In transgenic plants, the pin2/Gus construct showed low GUS activity and a weak wound response. However, in plants harboring the pin2/Act1 intron/Gus construct, wounding of a leaf dramatically increased the production of GUS. Moreover, the wound response is systemic because high levels of GUS activity were also found in non-wounded leaves that are adjacent to the wounded leaf.