Are At3G60030 and At2G47070 Lethal to Seed Development?

Jason Chai

HC70AL

What are my genes?At3G60030

At2G47070

Relevant Information of Genes

At3G60030:

- 3rd chromosome

- Opposite orientation

- 3717 bp long

- 928 amino acids

- 10 exons

- 9 introns

At2G47070:

- 2nd chromosome

- 5’ 3’ orientation

- 4173 bp long

- 882 amino acids

- 10 exons

- 9 introns

What Protein Does My Genes Encode?

At3G60030:- Squamosa promoter-

binding protein- SPL 12- Plant-specific

transcription factor- Related to flower

development

At2G47070:- Squamosa promoter-

binding protein- SPL 1- Plant-specific

transcription factor- Related to flower

development

Important Information of Protein

- The Arabidopsis thaliana Squamosa binding protein-like (SPL) gene family represents a group of structurally diverse genes encoding putative transcription factors found only in plants.

- The distinguishing characteristic is the SBP-box encoding a highly conserved protein domain of ~80 amino acids in length.

- This SBP-domain is responsible for the interaction with DNA.

- SBP-box genes play a role in plant development, mainly in the flowers.

SPL Family- SPL8 mutation - Affects pollen sac development in

Arabidopsis.- Anther at stage 9 to 10- Mutant only bears two pollen sacs

with few microspores.- Wildtype contains more pollen

sacs with free-floating microspores.

Figure 4. Light Microscopic Analysis of Developing spl8 Mutant Anthers.(A) Cross-section through spl8-1 mutant anthers comparable to stage 4 to 5 of wild-type anther development. spl8-1 mutant anthers often develop fewer than four pollen

sacs (arrowheads).(B) An spl8-1 mutant pollen sac at a higher magnification to illustrate the often-observed disturbed tissue differentiation. In particular, the outer secondary parietal cells may not undergo a further mitotic division and remain undifferentiated. Intermittently, in the developing outer secondary parietal layer, formation of presumptive endothecium and

middle layer cells caused by periclinal divisions (arrows) can be seen. Furthermore, the cells of the inner secondary parietal layer or tapetum are less well differentiated, reduced in number, and have an appearance closer to that of the sporogenous cells. Sporogenous cell number also is reduced compared with that in the wild type.

(C) The failure of spl8-1 sporogenous cells to undergo meiosis, as observed after stage 5 of wild-type anther development, and the subsequent decay of the pollen sac is revealed by the dense staining of their cytoplasm and the vacuolated tapetal cells.

(D) Somewhat oblique cross-section through a wild-type pollen sac at stage 5 to 6 of anther development just before meiosis. All cell layers are well differentiated, and dark-staining callose surrounds the meiocytes.

(E) Cross-section through an spl8-1 mutant anther comparable to stage 9 to 10 of wild-type anther development (cf. [F]). This anther bears two pollen sacs with some free microspores.

(F) Cross-section through a wild-type anther at stage 9 to 10. All pollen sacs are well developed and contain free vacuolated microspores with exine walls.ct, connective tissue; en, endothecium; ep, epidermis; g, gynoecium; mc, meiocyte; ml, middle layer; msp, microspore; osp, outer secondary parietal cell layer; p, petal; s,

sepal; sc, sporogenous cells; ta, tapetum; vc, vascular cells. (B) to (D) are at the same scale, as are (E) and (F). Bars = 25 µm in (A), 10 µm in (C), and 50 µm in (F).

What are the genotypes of Plant 1?

Expected size of WT: 1841 bp Total Homozygous mutant: 7

Expected size of Mutant: 620 bp Total Wildtype: 5

Total Heterozygous: 4

What are the Genotypes of Plant 2?

Plan

t 10

Plan

t 12

Plan

t 13

Plan

t 14

Plan

t 11

Plan

t 15

Plan

t 16

Plan

t 17

Plan

t 18

Plan

t 19

Plan

t 20

Plan

t 21

Plan

t 22

Plan

t 23

Plan

t 24

Wild

type

Posi

tive

Neg

ativ

e

Expected size of WT: 1514 bp Total Wildtype: 24

Where is At3G60030 active?

Expected size of RT-PCR: 452 bp

Where is At2G47070 active?

Expected size of RT-PCR: 369 bp

Are there any phenotypical differences?

Wildtype vs At3G60030

Cloning the Upstream of At3G60030

Cloning the Upstream of At2G47070

Special Thanks to the Officers in Charge

General: Dr. Bob Goldberg

Lieutenant General: Dr. Ahnthu Bui

Major General: Dr. Xingjun Wang

Brigadier Generals: Tomokazu Kawashima & Brandon Le

Colonels: Mike Gavino, Jonathan Russell, Ria Yagnik & Jessica Luke

Privates: Melissa, Thi, Matt, Jordan, Jennifer, Bekah, Brian, Jason, Daisy, & Heather