Kerckhoffs L H, Kelmenson P M, Schreuder M E, Kendrick C I, Kendrick R E, Hanhart C J, Koornneef M, Pratt L H, Cordonnier-Pratt M M
Department of Botany, University of Georgia, Athens 30602, USA.
Mol Gen Genet. 1999 Jul;261(6):901-7. doi: 10.1007/s004380051037.
The structure of the gene encoding the apoprotein of tomato phytochrome B2 (PHYB2) has been determined from genomic and cDNA sequences. The coding region is organized into four exons, like almost every other angiosperm phytochrome (phy). The deduced phyB2 apoprotein (PHYB2) consists of 1121 amino acids, with 82, 74 and 70% identity to tomato PHYB1, Arabidopsis PHYB, and Arabidopsis PHYD, respectively. In order to facilitate the identification of new mutants, we constructed a double mutant that is deficient in phyA and phyB1. When grown in white light, this mutant becomes only slightly taller than wild type and is similar in phenotype to the monogenic phyB1-deficient mutant. This double mutant has been used as the parent line for mutagenesis with gamma radiation. Several recessive mutants with long hypocotyls and reduced anthocyanin content were selected under white light and screened for mutations in PHYB2, PHYE and PHYF. Two of the triple-mutant lines, designated 55H and 70F, had elongated hypocotyls and fruit trusses, and pale immature fruits. Both belong to the same complementation group and both were found to have defects in PHYB2. Line 70F was found by Northern analysis to have a slightly larger PHYB2 transcript. Part or all of the intron between the second and third exons was found to be retained following RT-PCR of PHYB2 mRNA from line 70F. Three base substitutions were detected near the donor splice site for this intron, including a change from the consensus /GT to /GA at the 5' end of this intron. In every case, the C-terminal 164 amino acids of PHYB2 were replaced by 59 nonsense amino acids followed by a stop codon. Sequencing of PHYB2 from 55H revealed a single-nucleotide deletion near the end of the third exon, resulting in one incorrect codon followed immediately by a stop codon. The predicted mutant apoprotein in 55H is 90 residues shorter than wild-type PHYB2.
已通过基因组和cDNA序列确定了番茄光敏色素B2（PHYB2）脱辅基蛋白的编码基因结构。编码区像几乎所有其他被子植物光敏色素（phy）一样，由四个外显子组成。推导的phyB2脱辅基蛋白（PHYB2）由1121个氨基酸组成，分别与番茄PHYB1、拟南芥PHYB和拟南芥PHYD有82%、74%和70%的同源性。为了便于鉴定新的突变体，我们构建了phyA和phyB1缺失的双突变体。在白光下生长时，该突变体仅比野生型略高，并且在表型上与单基因phyB1缺失突变体相似。这个双突变体已被用作γ辐射诱变的亲本系。在白光下选择了几个下胚轴长且花青素含量降低的隐性突变体，并筛选了PHYB2、PHYE和PHYF中的突变。其中两个三突变体系，命名为55H和70F，具有伸长的下胚轴和果穗，以及浅色的未成熟果实。两者属于同一互补群，并且都被发现存在PHYB2缺陷。通过Northern分析发现70F系的PHYB2转录本略大。在对70F系的PHYB2 mRNA进行RT-PCR后，发现第二和第三外显子之间的部分或全部内含子被保留。在该内含子的供体剪接位点附近检测到三个碱基替换，包括该内含子5'端从共有序列/GT变为/GA。在每种情况下，PHYB2的C端164个氨基酸被59个无义氨基酸取代，随后是一个终止密码子。对55H中PHYB2的测序揭示了第三外显子末端附近的一个单核苷酸缺失，导致紧接着一个终止密码子出现一个错误密码子。55H中预测的突变脱辅基蛋白比野生型PHYB2短90个残基。
