Methods and compositions for selective regulation of protein expression

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The invention provides methods and compositions for selectively suppressing the expression of a recombinant protein in a male reproductive tissue of a transgenic plant. The invention also provides methods and compositions for inducing male sterility in a transgenic plant. Plants, plant cells, plant parts, seeds, and commodity products including such compositions are aspects of the invention.

The invention relates generally to methods of selectively suppressing recombinant protein expression in the male reproductive tissue of transgenic plants, recombinant DNA constructs useful in such methods, as well as transgenic plants, cells, and seeds containing such recombinant DNA constructs. The recombinant DNA constructs and the transgenic plants, cells, and seeds containing such constructs provide a greatly improved way to use herbicides for inducing male sterility in transgenic plants for the production of hybrid seed.

In one aspect, the invention provides a recombinant DNA construct that includes a protein-coding sequence encoding a recombinant protein and a male tissue- specific siRNA (mts-siRNA) element operably linked to the protein-coding sequence. In one embodiment, the mts-siRNA element is included within the 3' untranslated region of the protein-coding sequence. In another embodiment, the mts-siRNA element is located between the protein-coding sequence and a polyadenylation sequence which is part of a 3' untranslated region. In another embodiment, the mts-siRNA element includes at least one mts-siRNA sequence. In another embodiment, the mts-siRNA element includes at least one mts-siRNA sequence selected from the group consisting of SEQ ID NO: 1-56 and 105- 149. In another embodiment, the mts-siRNA element is selected from the group consisting of SEQ ID NO: 57-94 and 96- 104. In another embodiment, the expression of the recombinant protein in a transgenic plant confers at least vegetative herbicide tolerance to the plant. In another embodiment, the recombinant protein is a glyphosate-tolerant EPSPS.

Another aspect of the invention provides a method of making a recombinant DNA construct including identifying an mts-siRNA element including at least one mts- siRNA sequence and operably linking the mts-siRNA element to a protein-coding sequence, for instance a DNA sequence encoding a recombinant protein. In one embodiment, the mts- siRNA element includes at least one mts-siRNA sequence selected from the group consisting of SEQ ID NO: 1-56 and 105- 149or is at least one mts-siRNA element selected from the group consisting of SEQ ID NO: 57-94 and 96- 104. In another embodiment, the mts-siRNA element is tassel- specific.

In a further aspect, the invention provides a transgenic plant including a recombinant DNA construct of the invention, as well as a seed, cell, or part of the transgenic plant. In one embodiment, the plant is a monocotyledonous plant. In another embodiment, the plant is a maize (Zea mays) plant. In a further aspect, the invention also provides a method of selectively suppressing the expression of a recombinant protein in a male reproductive tissue of a transgenic plant by expressing in the transgenic plant a recombinant DNA construct that includes a protein-coding sequence operably linked to a DNA sequence including an mts- siRNA element. In one embodiment, the mts-siRNA element includes at least one mts- siRNA sequence. In another embodiment, the male reproductive tissue is a tassel of a maize plant. In another embodiment, the mts-siRNA element includes at least one mts-siRNA sequence selected from the group consisting of SEQ ID NO: 1-56 and 105-149. In another embodiment, the mts-siRNA element is at least one element selected from the group consisting of SEQ ID NO: 57-94 and 96-104. In another embodiment, the expression of the recombinant protein in a transgenic plant confers at least vegetative herbicide tolerance to the plant. In another embodiment, the recombinant protein is a glyphosate-tolerant EPSPS.

The invention also provides a method of inducing male sterility in a transgenic plant, including the step of applying herbicide to a transgenic plant that has in its genome a recombinant DNA construct comprising a protein-coding sequence operably linked to a DNA sequence including an mts-siRNA element that confers at least vegetative herbicide tolerance to the transgenic plant, wherein the herbicide is applied during the development of the male reproductive tissue of the transgenic plant thereby inducing male-sterility in the transgenic plant. In one embodiment, the transgenic plant is a maize plant. In another embodiment, the herbicide application prevents at least pollen shed or anther extrusion in the treated transgenic plant. In another embodiment, the development stage of the male reproductive tissue during which herbicide is applied is a stage selected from the group consisting of the V4, V5, V6, V7, V8, V9, V10, VI 1, V12, V13, and V14 stage of maize plant development. In another embodiment, the herbicide is selected from the group consisting of acetyl coenzyme A carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors, 4-hydroxyphenyl dioxygenase (HPPD) inhibitors, 5-enolypyruvyl shikimate 3- phosphate synthase (EPSPS) inhibitors, glutamine synthetase (GS) inhibitors, and synthetic auxins. In another embodiment, the herbicide is glyphosate and the recombinant protein is a glyphosate-tolerant EPSPS.

The invention also provides a method of producing hybrid seed including applying an effective amount of an herbicide to a transgenic plant including in its genome a recombinant DNA construct comprising a protein-coding sequence operably linked to a DNA sequence including an mts-siRNA element, wherein the herbicide is applied during the development of the male reproductive tissue of the transgenic plant thereby inducing male sterility in the transgenic plant; fertilizing the transgenic plant with pollen from a second plant; and harvesting hybrid seed from the transgenic plant. In one embodiment, the transgenic plant is maize. An effective amount of an herbicide is a dose of herbicide sufficient to render a transgenic plant comprising a recombinant DNA construct of the invention male sterile (an effective dose). In another embodiment, the herbicide is glyphosate and the recombinant protein is a glyphosate-tolerant EPSPS. In another embodiment, the glyphosate is applied during the development at an effective dose of about 0.125 pounds acid equivalent per acre to about 8 pounds acid equivalent per acre. Other specific embodiments of the invention are disclosed in the following detailed description. Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer, element, or step or group of integers, elements, or steps, but not the exclusion of any other integer, element, or step or group of integers, elements, or steps.